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Review of long noncoding RNAs and chromosome structure

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Review of long noncoding RNAs and chromosome structure

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Content 1

Review of Long noncoding RNAs and chromosome structure

by
Zhenyu Peng

______________________________________________________________________________________________

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry & Molecular Biology)

August 2017

Copyright 2017 Zhenyu Peng
2

Table of Contents

Abstract------------------------------------------------------------------------------------------------------------3
Introduction------------------------------------------------------------------------------------------------------3
LncRNA and intra-chromosomal looping-----------------------------------------------------------------4
Enhancer RNA (eRNA) Driven Enhancer-Promoter Interaction--------------------------------------------------------------4
RNA-a Driven Enhancer-Promoter Interaction-----------------------------------------------------------------------------------6
LncRNA and inter-chromosomal interactions-----------------------------------------------------------8
LncRNA and Nucleosome positioning---------------------------------------------------------------------10
ATP-dependent Remodeler Recruitment-----------------------------------------------------------------------------------------10
Impairing the binding of Remodeler-----------------------------------------------------------------------------------------------12
Direct Nucleosome positioning through transcription------------------------------------------------------------------------13
Genetic Discovery of LncRNA-------------------------------------------------------------------------------14
Genetic Characterization of LncRNA----------------------------------------------------------------------16
Useful Techniques in the study of LncRNA and Chromosome Structure------------------------19
Example Workflow for a study of LncRNA and Chromosome Structure-------------------------21
Reference---------------------------------------------------------------------------------------------------------23

3

Abstract
Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via
DNA methylation and posttranslational histone modifications is a well-documented function of lncRNA in plants, fungi,
and animals. In this review, we will discuss how LncRNA can also control genome activity by affecting chromosome
structure, including DNA looping and nucleosome positioning, with examples across eukaryotic kingdom, and will
introduce the history and methods of LncRNA research. We explain the mechanisms of lncRNA-controlled chromatin
remodeling and the implications of the functional interplay between noncoding transcription and several different
chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin
modifications and structure.

Introduction
LncRNA, or Long Non-coding RNA, are typically defined as RNA that is longer than 100 nt, but without apparent protein
coding potential [1]. It consists a majority part of genome transcripts. In decades, scientists tried to find the function of
these non-protein coding RNA, but with little success, some may just consider them ‘transcription noise’ [2]. But in
recent year, with the development of technology and further research in post-transcript modification, more and more
evidences show that LncRNA might play a very important part in post-transcript modifications, Including DNA
methylation, post-translational histone modifications, and DNA structure modification [3-5].
However, the definition of lncRNA is still controversial. In fact, up to 90% of the early reports of RNA-producing
eukaryotic genomes did not provide much evidence that lncRNA was functional [6], indicating that many RNAs
generated outside the coding region originated from transcriptional noise or artifacts in sensitive detection methods.
Thus, if there is at least a functional evidence that meets the "causal character" criteria, the RNA can only be classified as
lncRNA [7]. The definition of lncRNA also requires that it be independent of its coding potential. This is important
because it is assumed that non-coding RNA may encode a polypeptide that messenger RNA may have a function
independent of the coding protein [8]. In addition, various lncRNAs generally do not have a common evolutionary origin,
a biological function or a molecular mechanism. Therefore, the term "lncRNA" should be used with caution to avoid
suggesting mechanical, functional or evolutionary protection [9].
There are many lncRNAs with various documented functions that are growing rapidly. It has been shown that lncRNA
controls chromatin levels across the eukaryotic kingdom genome activity [2, 8]. For example, mammalian Xist RNA
controls chromatin-mediated X chromosome inactivation [9], while lncRNA HOTAIR recruit chromatin modifying
enzymes and mediates mammalian specific target loci histone modifications [10]. Recent study reported that LncRNA
can also function as a modulator to the chromosome structure level, including chromosome looping and nucleosome
positioning [2,8].
eRNA, or Enhancer RNA, is the LncRNA that transcribed from the known enhancer site. eRNA is mostly un-
polyadenylated nascent RNA molecules with low copy number in nucleus [10]. Studies has shown that many eRNA cells
will plays an essential role in the regulation function of the enhancer that it is transcribed from. There are also enhancer-
like RNA, or some will call them activating RNA(RNA-a) [11], that act in an enhancer-like way, regulating nearby genes in
cis. Typically, RNA-a is mature RNA molecule that is polyadenylated and spliced into different isoform like mRNA, and is
reported accumulating in the site of its interaction in high copy number [12]. Recently, evidences suggest that these two
subclasses of LncRNA are all involved in the regulation of gene expression by modulating chromosome structure.
4

Chromosome looping refers to the change in the high-order structure of chromosome [13]. Recent studies indicate that
chromosome looping is essential in many gene regulation processes [14]. For instance, the enhancer-promoter looping
formed between promoters and distal enhancers is one important way to gain spatial proximal and activate promoter by
distal enhancers. CTCF, Mediator Complex and Cohesin are reported to be key factors in the establishing and
maintaining of chromosomal looping [15]. Different kinds of LncRNA has been reported to interact with CTCF and
Mediator, thus participate in the forming and stabilization of chromosome looping. Evidence also suggests that there
could be structure changes between different chromosomes with the binding of LncRNA, forming spatial proximity
among locus on different chromosome [16]. But the mechanism of this multi-chromosome structure change is still not
well-studied.
Nucleosomes are fundamental unit of chromatin whereby DNA is wrapped around histone octamers. Tight interaction
with histone cores can strongly affect DNA accessibility [17]. Moreover, nucleosome positioning is a critical factor in
controlling gene expression, and it is determined by a combination of local DNA features and active remodeling. Recent
studies suggest that LncRNA could modulate nucleosome positioning by recruitment of ATP-dependent remodeler and
transcription-mediated nucleosome stabilization [18].
In this review, we will introduce the history and methods of LncRNA research, discuss how LncRNA can control genome
activity by affecting chromosome structure, including DNA looping and nucleosome positioning, with examples across
eukaryotic kingdom.

LncRNA and intra-chromosomal looping
Enhancer RNA (eRNA) Driven Enhancer-Promoter Interaction
Enhancer is a well-known DNA regulatory element that plays a central role in gene regulation. Previous studies indicated
enhancer function involved the recruitment of transcription factors to promote the detangling of repressed chromatin
and facilitate the assembly of transcriptional machinery on target genes [19]. Enhancer RNA (eRNA) are a class of
LncRNA that is transcript from enhancer sequences, typically non-polyadenylated nascent RNA with very low copy
number [20]. It is still unclear whether eRNAs are just by-product of enhancer activity or whether they have their
function in gene regulation. Recent studies show that eRNA may play an important role in the DNA looping enhancer-
promoter interaction [19].
The Human DHRS4 gene is an NADPH-dependent enzyme(NRDR) encoding gene located on chromosome 14q11.2 [21].
There are two homologous downstream genes, DHRS4L1 and DHRS4L2, which could form a gene cluster with DHR4 [22].
Previously, scientists have already found that the expression of DHRS4 could be regulated by a NAT (nature antisense
transcript) named DHRS4-AS1 [23]. DHRS4L1 and DHRS4L2 is highly sequence homologous with DHRS4 and all contains
the same promoter sequence of DHRS4-AS1, but no evidence shows that similar NAT could be produced from the
antisense template of these two genes [24]. This suggests that there could be an enhancer-promoter interaction in the
transcript of DHRS4-AS1 so that only the enhancer-interacted promoter of DHRS4 could be activated, but not those of
DHRS4L1 and DHRS4L2. Active enhancers are typically marked by high levels of H3K4me1, low levels of H3K4me3, and
high levels of H3K27ac. Based on these histone modification features, scientists could identify an enhancer located
13.8kb downstream of the DHRS4-AS1 TSS. It is then confirmed that this enhancer can directly interact with DHRS4-AS1
promoter by chromosome looping by using 3-C (chromosome Conformation Capture) technique. Further evidence
shows that this enhancer function by produce eRNA, named AS1eRNA, that can mediate chromatin looping between
enhancer and the DHRS4-AS1 promoter, thus enhance the transcript of DHRS4-AS1 NAT [24].
5

RNA Pol II and the transcriptional coactivator p300 is key element in maintaining the chromatin looping by occupy both
enhancer and target promoter regions. In HepG2 cells, the depletion of DHRS4-AS1 lead to a reduce in the binding of
RNA Pol II and p300 with both promoter and enhancer. Suggests that DHRS4-AS1 may function through mediate the
long-range chromatin interaction with RNA Pol II and p300 between the As1 enhancer and the DHRs4-AS1 promoter. In
the working model proposed in this study, AS1eRNA may have binding affinity to Pol II and p300 that occupy promoter
region and Pol II and p300 that occupy enhancer region respectively, thus mediating the spatial proximal of the
promoter and enhancer [24].
In another study about estrogen-receptor α (ER- α), scientists report a global increase in eRNA transcription activity on
enhancers that adjacent to E2 upregulated genes after E2 binding to ER- α on enhancers. This indicates that there should
be connection between the activation of these E2 upregulated genes, and the increasing transcription of the adjacent
enhancers. Scientists then select ten highly-upregulated transcripts for further study. Using RIP-Seq and RIP-qPCR, they
find that there’s interaction between Cohesin and eRNAs. Knock-down of these eRNA may result in a decrease in
Cohesin recruitment. Depletion of some of the eRNAs including SMC3 and Rad21 would cause loss of promoter-
enhancer interactions and block the coding gene induced by E2. This suggests that eRNA could participate in
chromosome looping by interact with the key factor cohesin, thus enhance the interaction between promoter and
enhancer [25].
In both studies, when the enhancer is activated by ligand or other pathway, the transcription of enhancer RNA will be
activated. The Nascent eRNA transcript will stay with the transcription complex, and then form interaction with other
factors like p300 or Cohesin to mediate the chromosome looping in a Cis-pattern. (Fig.1) [26] Precise mechanism of how
eRNA mediate the looping, whether these eRNA interact with key factors CTCF and Mediator remain a question.

6

Figure.1 [26] Possible model for the mechanism of eRNA in mediating chromosome looping. In:
Shibayama Y, Fanucchi S, Magagula L, Mhlanga MM. lncRNA and gene looping: what’s the connection? Transcription.
2014;5(3):e28658. doi:10.4161/trns.28658.

RNA-a Driven Enhancer-Promoter Interaction
MYC gene is an oncogene located in Human chromosome 8 [27]. The interaction between MYC gene and the gene
dessert located on 8q24 region is tissue-specific in cancer cells including human breast, prostate and colorectal cancer.
In Colorectal Cancer(CRC), a well-characterized loop is between MYC and MYC-335, which is an enhancer 335 kb
upstream of MYC gene [28]. Human 8q24 gene dessert region has reported to express different kinds of lncRNAs in
different cancer cell-lines [29]. One of these tissue- specific LncRNA expressed in CRC is called CCAT1-L (Colorectal
Cancer Associated Transcript 1, long isoform), which is 5200nt length and transcribed 515 kb upstream of MYC (MYC-
515) [30]. In the study, scientists found that MYC-515 can form loop with both MYC-335 and MYC gene(Fig.2) [31].
Knockdown of CCAT1-L will reduce the expression level of MYC gene, indicating LncRNA CCAT1-L plays an important role
in the regulation of MYC genes. Knockdown of CCAT1-L would significant reduce the interaction between not only MYC-
515 and MYC-335, but also MYC-335 and MYC genes. Indicates that CCAT1-L as well as the spatial proximal of MYC-515
Cohesin
7

are essential part of the interaction between enhancer MYC-335 and promoter of MYC genes. By DNA FISH visualization,
CCAT1-L shows strong colocalization with all three loci, MYC, MYC-335 and MYC-515.Different from eRNA that is nascent
and non-polyadenylated, functioning when still connected to transcript factors, CCAT1-L LncRNA is fully transcribed and
polyadenylated, accumulated in the loci of interaction [31].
Further study indicates that the looping of MYC promoter and MYC-335, MYC-515 is CTCF-mediated by ChIP-Seq study.
The Knockdown of CTCF will disrupt the looping, dramatically reduce the interaction of MYC promoter and MYC-335,
MYC-515. Importantly, the knockdown of CTCF will also decrease the transcription of CCAT1-L, indicates there may be a
positive regulatory network of MYC including CTCF and CCAT1-L. By using biotin-labeled RNA pulled-down assays and
RNA immunoprecipitation, scientists confirm that there is direct interaction between CTCF and CCAT1-L. Knockdown of
CCAT1-L led to a modest reduction of CTCF binding to chromatin at their occupied chromatin sites in loop-forming
regions at the MYC. This suggests that CCAT1-L lncRNA may act to locally concentrate CTCF or allosterically modify CTCF
binding to chromatin to maintain the chromatin looping in the 8q24 region surrounding the MYC locus in CRC cancers
[31].
As mentioned before, MYC gene is an oncogene in many different cancers, and the 8q24 gene dessert region reported to
express several different LncRNA in different cancer [32]. Recent study also suggests that this 8q24 region could be a
cancer-specific super-enhancer that only become enhancer-like in cancer cells. (High H3K27ac, high H3K4me1, low
H3K4me3) [33]. Although the genomic locus spans up to 150kb, while a typical enhancer is about ~1.5kb in length. How
is this super-enhancer behave in other cancer? Would the other cell-specific LncRNA expressed in this region also have a
similar function to CCAT1-L, by mediating the chromosome looping between MYC gene and its enhancer? These
questions are worth considering, to find the mechanism behind 8q24 gene dessert region in regulating MYC-genes, and
even the other super-enhancer region across human genome.

8

Figure 2. The spatial relation of MYC, MYC-335 and MYC-515

LncRNA and inter-chromosomal interactions
Long-range chromosome interactions occur not only on the same chromosome, but also among different loci across
different chromosomes. Recently, LncRNA Firre (Functional intergenic repeat RNA element) has been reported to act as
an important part in the organization of multiple chromosomes to establish a nuclear domain [34]. Firre is a 5.8kb
LncRNA transcribed on X chromosome containing 156-bp repeating sequence that form secondary structure [35].
Previous study showed that Firre plays a role in adipogenesis. By performing RNA Antisense Purification (RAP), scientists
have observed a 5Mb domain of Firre localized around the Firre locus [34]. Strikingly, they also observed an enrichment
of Firre on chromosome 2, 9, 15, 17, overlapping known protein-coding genes Slc25a12, Ypel4, Eef1a1, Atf4,
and Ppp1r10. 4 out of 5 of these genes are previously identified regulators in adipogenesis [36-38]. These observations
MYC-335 MYC-515
MYC
CCAT1-L
loop2
loop1
CCAT1-L RNA
loop2
loop1
9

suggest two possible models. One model is that Firre could be shuttled from its transcription site to these sites on other
chromosomes. The other model would be, the focal localization of Firre to its own genomic locus could serve as a
regional organizing factor to bring the trans-interacting sites into the three-dimensional proximity of the Firre locus on
the X-chromosome. In further study, by using single molecule RNA co-FISH, scientists found that these Firre localized
locus are spatial proximal. Moreover, the knockdown of Firre will result in the loss of co-localize of these genes. This
finding suggests that the second model may be correct [34]. Firre may play an essential role in the either the establishing
or the maintaining of a high-order chromosomal architecture that bring genes located on different chromosome spatial
proximal. But how this topological organization may affect the regulation of these involved genes required further
research.
In another study about LncRNA CUDR (Cancer Upregulated Drug Resistant) in human liver stem cells, scientists found
similar function [39]. CUDR is a LncRNA highly-expressed in many cancer cell-lines [40]. The overexpression of CUDR in
human liver stem cell will lead to malignant transformation. In the study, CUDR was found to participate in the
promoter-enhancer looping of β-catenin, by interacting with CTCF [39]. But in this case, CUDR and β-catenin are not on
the same chromosome. CUDR is on chromosome 19 and β-catenin gene CTNNB1 in located on chromosome 3. This
means that CUDR, unlike CCAT1-L, is not accumulated on the transcription locus and mediating looping on the same
chromosome in an enhancer-like way, but participating a distal looping that is on another chromosome, acting as a
trans-element. The overexpression of CUDR will result in an increase interplay of CTCF and CUDR, an increase in the
interaction between CTCF and β-catenin enhancer and promoter, an increase in the Pol II and p300 recruited, and finally
an increase in the expression level of β-catenin [39]. But in this study, it is not clear about the relation between CUDR
and other factors in the enhancer-promoter looping of β-catenin besides CTCF. Is the binding of CUDR and CTCF a global
interaction that occur on CTCF in different locus? Or is it a locus-specific interaction that only occurs on the β-catenin
locus? If this is a global interaction, does it means that CUCR can participate in many different chromosome looping? If it
is locus-specific, then how is CUCR guided to the site? These question remains unanswered.
Interestingly, CUCR does not only function as a factor in DNA looping, but also through methylation inhibitor activity. In
the same study, scientists find that CUDR can induce the expression of HULC (long non-coding RNA highly upregulated in
liver cancer) [39]. As the name suggests, HULC is a kind of LncRNA that is highly upregulated in human liver cancer cells
[41]. Overexpression of CUCR will reduce the methylation of HULC promoter, thus increase the expression level of HULC,
which would further participate in the hepatocarcinogenesis (fig.3) [39]
10

Figure.3 [39] The Function of CUDR in the regulating of hepatocarcinogenesis. In:
Gui X, Li H, Li T, Pu H, Lu D. Long Noncoding RNA CUDR Regulates HULC and β-Catenin to Govern Human Liver Stem Cell
Malignant Differentiation.Molecular Therapy. 2015;23(12):1843-1853. doi:10.1038/mt.2015.166.

LncRNA and Nucleosome positioning
ATP-dependent Remodeler Recruitment
Nucleosomes present a major obstacle for the binding of sequence-specific DNA-binding factors, the interaction of
positively charged histone tails with DNA and the masking of DNA binding sites that face in towards the histone octamer
11

surface [42]. All the DNA-dependent processes including transcription, replication, repair and recombination are related
to the positioning of nucleosomes on the regulation site [43]. ATP-dependent chromatin remodeling complexes, which
use ATP to slide, replace or evict histone on nucleosome, is a key regulator in the nucleosome positioning and chromatin
structure [44]. In human and other mammalian cells, the role of small non-coding RNA in the regulation and targeting of
the ATP-dependent chromatin remodeling complex is well-discussed, but not the LncRNA. In the meanwhile, recent
studies show that LncRNA is an important factor in the nucleosome positioning in plants [45].
RNA polymerase V (Pol V) is a multi-subunit plant specific RNA polymerase found in nucleus [46]. In Arabidopsis, lncRNA
produced by Pol V serves as a binding scaffold for several RNA-binding proteins including INVOLVED IN DE NOVO 2
(IDN2) [47]. This protein was discovered in forward genetic screens and was shown to be required for RNA Direct DNA
Methylation (RdDM). IDN2 physically interacts with SWI3B, a core subunit of the most well studied ATP-dependent
chromatin remodeling complex, the SWI/SNF complex [48]. The SWI/SNF complex regulates gene transcription as a
multi-protein system that physically move nucleosomes at gene promoters. This interaction guides the SWI/SNF
complex to loci transcribed by Pol V, where specific nucleosomes are stabilized. This way, lncRNA produced by Pol V in
Arabidopsis is involved in active nucleosome positioning by binding IDN2 and recruiting SWI/SWF complex to the loci of
Pol V transcription [49].
Recruitment of the SWI / SNF complex to the RdDM-targeted site may also involve additional lncRNA binding proteins. It
has been shown that the binding of IDN2 to lncRNA requires a previously present ARGONAUTE4 (AGO4), which is the
main Argonaute involved in RdDM in Arabidopsis [49]. AGO4 introduces siRNA that can provide sequence specificity to a
genomic region by base pairing between siRNA and lncRNA. Since SWI3B was recruited by IDN2, the combination of SWI
/ SNF and RdDM targets may require AGO4 and siRNA. Another lncRNA binding protein involved in RdDM is the inhibitor
type 5-LIKE (SPT5L), which binds to silenced loci parallel to AGO4. Although the function of SPT5L and its effect on the
binding of IDN2 to lncRNA is still unknown, it is required for transcriptional silencing at least on a subset of the RdDM
target. This suggests that SPT5L may also participate in SWI / SNF recruiting chromatin. Similarly, maize homologs that
have been shown to be RNA-dependent RNA polymerase required for siRNA production affect nucleosome localization
on a specific locus. Although there is no indication that RdDM-mediated recruitment of SWI / SNF in maize, this further
indicates that additional RdDM components are involved in nucleosome positioning [49].
This way, lncRNA produced by Pol V in Arabidopsis is involved in active nucleosome positioning by binding IDN2 and
recruiting SWI/SWF complex to the loci of Pol V transcription. The binding of AGO4 and SPT5L with the Pol V transcribed
LncRNA may also affect the recruitment of SWI/SWF complex. The SWI/SNF complex positions nucleosomes, which
affect Pol II transcription by facilitating DNA methylation and/or restricting protein access to DNA. (Fig.4) [45]
Similar mechanisms exist in yeast. In S. pombe, pericentromeric and other heterochromatic regions are transcribed into
LncRNAs, and these LncRNAs are bound by Seb1, a homolog of the conserved RNA binding protein Nrd1 [50]. Seb1
recruited the SHREC complex, which contains the putative Snf2 chromatin remodeler Mit1, which is necessary for proper
nucleosome positioning [51-53]. SHREC eliminates the nucleosome-free regions and establish histone H3 lysine 9
dimethylation(H3K9me2) [51]. Thus, the transcription initiation site may become inaccessible, and Pol II association may
be inhibited. These results together show that in the fission yeast, lncRNA regulates the location of nucleosomes by
recruiting ATP-dependent chromatin remodeling factors[54]. This mechanism is similar to that in plant RdDM, where
chromatin recombination is raised by heterochromatic lncRNA. An important difference is that the SHREC recruitment
does not involve siRNA or Argonaute, which seems to work in parallel with RNAi. Although several evidences suggest
that lncRNAs control the positioning of nucleosomes in various organisms, whether it is the main mechanism of the
recruitment of chromatin remodelers causing this phenomenon remain unknown [45].

12

Figure.4 [45] Model of LncRNA ATP-dependent Remodeler Recruitment. In:
Böhmdorfer G, Wierzbicki AT. Control of chromatin structure by long noncoding RNA. Trends in cell biology.
2015;25(10):623-632. doi:10.1016/j.tcb.2015.07.002.

Impairing the binding of Remodeler
Previously, we discussed the role of LncRNA in the recruitment of ATP-dependent remodeler to the promoter nearby the
transcription site of LncRNA in cis. In another study, scientists report that LncRNA could also act as an inhibitor in the
recruitment of remodeler to promoter in an in trans way.
In this study, scientists characterize a novel LncRNA SChLAP1 (Second Chromosome Locus Associated with Prostate-1),
which overexpressed in a subset of prostate cancer [55]. In vitro and in vivo experiment indicates that this LncRNA
should pay a critical role in cancer cell invasiveness and metastasis. When performing the knockdown of SNF5(also
13

known as SMARCB1), an essential subunit of SWI/SWF [56], facilitating SWI/SWF binding to histone proteins, it shows
opposite effect on the expression level of genes that also regulated by SChLAP1. This indicates that SChLAP1 functions
antagonistically to SWI/SWF. Mechanistically, the knockdown of SChLAP1 have no impact on the expression level of
SNF5, demonstrating that SChLAP1 is not acting through directly regulation of the expression of SWI/SWF, but in a post-
transcriptional way. Further using RIP assays for SNF5, scientists observed that SNF5 are co-immunoprecipitated with
SChLAP1 but no other LncRNAs. In a ChIP-Seq of SNF5, 6235 genome-wide binding sites were found for SNF5, highly
enriched for sites which are near gene promoters [55]. When SChLAP1 is overexpressed, a dramatic decrease of SNF5
binding is found in these 6235 sites, and when SChLAP1 is knockdown, an increase of binding is observed in these sites.
Overall, these data suggest that SChLAP1 may antagonize the function of SWI/SWF by disrupt the genomic binding
activity of this complex, thus impairing its ability to regulate gene expression. Unlike the recruitment function we
discussed formerly, which mostly function near its site of transcription, this SChLAP1 appear to function across genome
in trans. It will directly interact with the SWI/SWF complex, thereby decrease its ability of promoter binding.
Interestingly, in this study, the decrease of SWI/SWF binding by overexpressed SChLAP1 will results in a primarily
downregulate of genes nearby the binding site, thought the function of SWI/SWF complex is known as regulating the
gene expression in either direction[55].

Direct Nucleosome positioning through transcription
Transcription-mediated silencing, also referred to as ‘transcriptional interference’ (TI), is defined here as a case in which
the act of transcription of one gene can repress in cis the functional transcription of another gene [57]. The DNA in the
nucleus is organized into chromatin, and the tissue scaffold consists of nucleosomes, each with two copies of H3, H4,
H2A and H2B histones [58]. Nucleosomes can be densely packed, interfering with protein-DNA interactions or relaxation,
and promoting these interactions. The transcriptional process of RNAPII along the gene locus can directly influence
nucleosome positioning. Thus, lncRNA transcription can cause TI by depositing nucleosomes in a manner that is not
conducive to TF binding on the promoter or enhancer [45]. An example of this mechanism is the silencing of the yeast
SER3 pc gene by the transcription of LncRNA SRG1 [60]. The SRG1 transcription process free up the space for binding by
moving the pre-occupying nucleosome aside, increases the density of nucleosomes on overlapping SER3 promoters, thus
block the binding of TF on the SER3 promoter. The deletion of three transcriptional elongation factors
SPT16, SPT6, SPT2, which are associated with nucleosome positioning will abolish the silencing effect on SER3 without
termination the transcription of SRG1. In contrast, the depletion of epigenetic modifiers including histone
methyltransferase and DNA methylation factors doesn’t affect the silencing of SER3, which means that SRG1 doesn’t
function through a methylation modification pathway, but the nuclear positioning. These finding indicates that the
transcription process of SRG1 can directly change the density of nucleosome on the nearby promoter site of SER3 gene,
thereby block the binding of TF to the promoter, and silence the SER3 gene. Although the role of SRG1 LncRNA molecule
is not excluded in the silencing of SER3 gene, scientists suggest that the transcription process alone can explain the
silencing [60]. (Fig.5)
The transcriptional interference by nucleosome repositioning may be a general mechanism in yeast, because the RNAPII
elongation and chromatin organization factors responsible for SER3 silencing, are also known to be involved in the
suppression of transcription initiation from cryptic promoters within the body of actively transcribed genes. Since genes
controlling RNAPII elongation and chromatin organization are largely conserved, it is possible that lncRNAs could use
similar nucleosome repositioning silencing in mammals [61].
14

Figure.5 The Nucleosome positioning through transcription of LncRNA.

Genetic Discovery of LncRNA
At the late 20
th
century and the beginning of 21
st
century, with the development of ‘human genome project’ [62, 63],
scientists are eager to find out how many genes are there in human genome, and is it possible to explain the complexity
of different organisms by the sheer number of classic protein coding genes, and the splicing diversity. With the
automated Sanger sequencing application in 1990’s, scientists could access the mapping of expressed sequence
tags(ESTs) [64, 65] that demonstrate the fragments of genomic regions that were being actively transcribed. And thus,
lead to the study in the field of ‘transcriptome’. In 1996, scientists were able to find an intriguing new notion that many
‘genes’ were mapped in yet undefined regions of the human genome [65]. But because of the limitation of short
sequence reads of Sanger sequencing at that time, and an incomplete reference human genome to aligned ESTs, it was
remained elusive what these new ‘genes’ may encode.
Tiling Microarrays—In addition to sequencing advances, new technologies were to apply to de novo identification of
new genes, and to better understand the regulation of gene-expression. Tiling microarrays is one of the novel
technology, allowing the ability to survey on the scale of 20,000 gene or genomic loci. In the same time, in 1999 [66], the
first complete human chromosome sequence —the sequence of human chromosome 22 was released. With not much
novel protein-coding genes discovered, the combination of human genome and microarray technology identified a wide-
spread of non-coding RNA across human genome. In that time, scientists believe that at least half of the transcripts from
human genome would be non-coding. And some believe that this may just be transcript noise that will not have any
function at all [67].
Therefore, one of the results of the Human Genome Project is the discovery of many new RNA genes, but not new
protein genes. For example, the number of human miRNAs has increased rapidly from a few to nearly one thousand [68-
Access Block
15

70]. In fact, further advances in RNA sequencing, cDNA cloning, and microarray technology over the next decade have
led to efforts within the coalition to define all the transcription genes in the human genome. The conclusion is that most
genomes are transcribed. Although extensive transcription is observed throughout the genome, the identification of
functional RNA molecules is equivalent to finding a needle in a haystack. In fact, this extensive concept of transcription-
rich has become increasingly controversial [71-74].
Chromatin marks - the key clues to capture RNA genes come from chromatin, and all eukaryotic genes are present in the
DNA protein complex. With the whole genome sequence, chromatin immunoprecipitation followed by deep sequencing
(ChIP-seq) resulted in a genomic map of the chromatin structure known as the " epigenome " [75-77]. The large-scale
parallel sequencing and its modification of the histone-occupying DNA sites reveals many interesting genomic domains.
(K4-K36 domain) Gene promoter and histone H3 lysine 36 Trimethylation (H3K36me3) Histone H3 Lysine 4
Trimethylation (H3K4me3) occupied by the polymerase II transcription genes [78-80]. In a study, the entire mouse and
human genome were measured by several cell type chromatin markers, showing approximately 5,000 K4-K36 domains
representing lncRNA [81]. These lncRNAs have discrete gene loci, which are located in previously unrecognized
intergenic regions prior to the protein coding gene, and thus these RNAs are designated as large insert non-coding RNAs
(lincRNAs). Further analysis of these loci revealed a highly conserved promoter region that recruited key transcription
factors for binding and direct regulation. LincRNA shows sequence conservation throughout the evolution of intron or
untransformed gene sequences, further demonstrating its function [82-87].
RNA-seq – The appearance of deep sequencing technology has led to an unprecedented sequential cDNA sequences and
sequence performance known as RNA-Seq [88-92]. These methods have been combined with the computational method
allowing the resolution of the reconstructed single nucleotide transcript and its isoforms. These studies provide an
unbiased method to identify non-coding transcripts across different cell types and tissues [93].
In addition to the full-length reconstruction algorithm, there also appeared some other applications to be include in
RNA-Seq. For example, a method called "3-seq" targeting the polyadenylation tail of cDNA [94], using more affordable
short reads to quantitatively measure the abundance of transcripts. In addition, the method can use variants to
accurately map the 3 'end of the transcripts [95]. Recently, metabolic markers have been used to measure nascent
transcript mRNA, thus providing transcription of the polymerase and dynamic pause points of view. These and many
other emerging technologies are providing a deeper insight into the dynamic transcriptome [96].
Recent studies have estimated that gene-specific identification of different categories of large RNA using RNA
sequencing and abundance of transcription. For example, a recent study by comments from many sources and RNA
sequencing were combined to determine the human genome gene among 8000 large non-coding RNA (lincRNA). The
study revealed several lncRNA global properties, including that lncRNA tend to locate alongside regulators, identifying
thousands of orthologous lincRNAs between human and mouse rich expression patterns specific to tissue as well as
genetic qualities of lincRNA positioning hundreds of genes associated with the desert. By increasing the use of
sequencing depth, the length and read some of the first stage in the lncRNA characterization allowed on a global scale
[93].
By combining the above-described techniques, it is now possible to determine all the transcription trajectories (K4-K36
chromatin domains), as well as RNA products (RNA-SEQ) as the main structure of the precise map. The information that
these combinatorial layers are synergistic with chromatin - modified gene loci to identify the stable transcription of the
RNA sequenced and even allow a single low - abundance transcript that can be said to be known as transcriptional noise
detection. The chromatin of the additional information currently indicates a given locus (H3K4me3) and a transcriptional
unit (H3K36me3), thus ending in the mapped promoter promoter region of the 5’ and 3’ of the RNA transcript.
Progressive additions through additional layers of information processing procedures (eg, protection, potential coding
16

patterns and anatomical properties) are being carried out to identify lncRNA gene families. LncRNAs have been further
defined according to the anatomical properties of their gene loci. For example, antisense lncRNAs, known as overlapping
of protein-coding genes, are known as introns that lncRNAs encode genes in the intron of the protein gene, and the
overlap of lncRNA protein coding genes is known as transcript encoding and lincRNAs that are genes The genome is
completely in the space between the protein coding loci., Although, it is likely that many of these lncRNAs will share
similar function and mechanism.

Genetic Characterization of LncRNA
Excluding the potential of protein coding
The basic to determine LncRNA is whether it could translate into protein or not. But this could be a very difficult task.
Many studies try to evaluate lncRNA coding potential by translating each lncRNA in all 3 'frames and performing
homology queries (ie BLASTX) on large protein families and domain databases (ie, Swissprot and PFAM) [94]. This
information analysis are good predictors of protein coding ability, but may miss new evolutionary protein sequences or
very small open reading frames (<50 amino acids). To solve the previous problem, codon substitution frequency (CSF)
analysis has been used to determine whether the codon of the amino acid is preferentially conserved through evolution,
indicating the preservation of the protein coding potential [95]. CSF has been used in several studies as additional
information layers for determining coding potential. However, even if these two methods are combined, it is still
possible to miss a small open reading frame hidden in these long transcripts. Experimental methods such as ribosomal
assays, identified by ribosome binding and scanning of the putative RNA, provide a further understanding of those RNAs
that may encode small peptides. In addition, the method identifies the region occupied by the ribosome, thereby further
honing the potential translation region, which can be used as an accurate predictor of information input such as CSF and
BLASTX. Although some of the lncRNA may encode small peptides, we note that this does not preclude the potential
dual nature of lncRNA that acts through RNA and its protein products. This has been demonstrated by many mRNAs
containing regulatory non-coding RNA elements (p53, Sgrs, Oskar, VegT, etc.) [96-101].
Through co-expression to infer the effect of lncRNAs: by Guilt-by-Association
Mapping to thousands of lncRNA loci, the next challenge is to determine what is lncRNAs. Assume that the first step is to
use the lncRNA expression pattern to identify the specific cell type or biological process associated with each candidate
lncRNA. Some of the first expression studies of lncRNA identified lncRNAs that were highly expressed in certain brain
regions. In situ hybridization studies further confirmed that these expression patterns showed a fine expression pattern
in the specific sub-structures of mouse brain. A similar study in this group found that many lncRNAs that are closely
related to pluripotent transcription factors indicate that many lncRNAs may play a role in stem cell pluripotent
transcription networks [102].
Recently, information methods known as " Guilt-by-Association " allow global understanding of lncRNA and protein-
coding genes that are closely co-expressed and thus may be co-regulated. This method identifies gene coding genes and
pathways that are significantly associated with a given lncRNA using gene expression analysis. Thus, based on the known
function of the co-expressed protein-encoding gene, the hypothesis of the function of the candidate nucleotide and the
potential regulator is produced. In addition, this analysis reveals the "family" of lncRNA [103], based on what they do
and irrelevant. This method has been predicted by the different roles of lncRNA, from stem cell pluripotency to cancer.
For example, many lncRNAs that are closely related to p53 are induced in a p53-dependent manner, much more than
expected. These lncRNAs also enrich the p53 binding motif in their promoter. In addition, it was found that one of these
lncRNAs known as lincRNA-p21 associated with p53 was directly regulated by p53 followed by the formation of a nuclear
17

factor of lncRNA-RNP as a function of promoting p53-mediated global transcriptional repressor. Similarly, several
lncRNAs predicted to correlate with adipogenesis and pluripotency are most often considered necessary to maintain
these cell states.
Other expression-related analyzes reveal additional functional effects of lncRNAs. For example, recent studies have
analyzed lncRNA across more than 130 breast cancers, which include different grades of tumor and clinical information.
This study identified many lncRNAs that were specifically up-regulated or down-regulated in tumor subtypes. For
example, it was determined that lncRNA, called HOTAIR [104], encoded in the HOXC cluster was a strong predictor of
breast cancer metastasis. In fact, HOTAIR's mandatory expression is sufficient to promote breast cancer metastasis. A
more comprehensive expression of the lncRNA in the protein-overlapping gene promoter region identified many
lncRNAs associated with cell cycle regulation. This leads to a functional representation of lncRNA called PANDA, which
plays a key role in inhibiting p53-mediated apoptosis. The " Guilt-by-Association " approach is universally applicable to
any biological system. For example, the telomere-encoding lncRNA family of P. falciprum was identified by its phase-
specific coexpression with the important virulence transcription factor of PfsiP2 [105].
These and other related studies have begun to determine the specific role of lncRNA in global transcriptional regulation.
Honing in lncRNAs in lncRNA-related pathways identified by hypothesis-driven experiments. However, lncRNA
transcriptional regulation and functional full range is far from understood. To learn more about the global regulatory
role of lncRNAs, it is necessary to perform functional experiments with integrated gain or loss.
High throughput loss of function by RNA inference
A recent study performed a lost-of-function study across Most of the long intergenic non-coding RNA (lincRNA)
expressed in mouse embryonic stem cells (ESC) [106]. The authors show that knockout lincRNA has a significant effect
on gene expression patterns, comparable to the known knockdown of ESC regulators. Interestingly, this global
fluorescent screen determines that lincRNA mainly affects gene expression in transcription. Perhaps more importantly,
in the maintenance of multi-energy state, found dozens of lincRNA functionally required. Further studies on the
molecular circuit of the ESC suggest that the lincRNA gene is regulated by a key transcription factor, and that the lincRNA
transcripts physically bind multiple chromatin to influence the shared gene expression program. This study provides a
first glimpse of the global lincRNA functional properties, and highlights their key role in controlling the ESC state of the
circuit. (Fig.6)

18

Figure.6 Determination of function LncRNA

RNA-Seq
Microarray
Chromatin
marks
Coding Potential
CSF PFAM
Guilt-by- Association
Lost of Function
Mechanism
Function
LncRNA
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Useful Techniques in the study of LncRNA and Chromosome Structure
ChIP-Seq
ChIP sequencing, also known as ChIP-seq, is a method for analyzing the interaction of proteins with DNA. ChIP-seq
combines chromatin immunoprecipitation (ChIP) with large-scale parallel DNA sequencing to identify DNA-associated
protein binding sites. Used to accurately map the global binding site to obtain any protein of interest. ChIP-seq may be
used to determine how transcription factors and other chromatin-related proteins affect phenotypic effects. It is
indispensable to determine how proteins interact with DNA to regulate gene expression. This is an epigenetic
information that is complementary to genotype and expression analysis. ChIP-seq technology is currently considered an
alternative to the ChIP chip that requires hybrid arrays. This is some positive because the array is limited to a fixed
number of collisions. In contrast, sorting is considered to have a small deviation, although sequence alignment of
different sequencing techniques has not yet been fully understood [107].
Specific DNA loci that interact directly with transcription factors and other proteins can be isolated by chromatin
immunoprecipitation. ChIP produces a library of target DNA sites that bind to the protein of interest in vivo. Many
parallel sequences analysis were used in conjunction with the whole genome sequence database to analyze the pattern
of interaction of DNA or any apparent genetic chromatin modification. This can be applied to encodeable proteins and
modifications, such as transcription factors, polymerases and transcription mechanisms, structural proteins, protein
modifications and DNA modifications. As an alternative to specific antibody dependencies, different methods have been
developed to find supersets of the active regulatory regions of all nucleosome deletions or nucleosomal destruction in
the genome, such as DNase-Seq and FAIRE-Seq [108].
RIP-Seq
RIP is an antibody-based technique for mapping in vivo RNA-protein interactions. Interested RNA binding proteins (RBP)
are Transact detected by real-time PCR, microarray or sequencing. When we begin to realize that interest in RNA-protein
interactions is booming, the role of RNA in it is not only in mature processes such as transcription, splicing and
translation, but also in newer fields such as RNA interference and non-coding RNA of gene regulation [109].
ChIRP
The chromatin isolation by RNA purification (ChIRP) is a strategy for mapping the full-length genome length at high
resolution based on the affinity of the target lncRNA: chromatin complex by tiling antisense oligonucleotides and
capture, and then on the antisense oligomer to produce genomic binding sites of the spectrum resolution of up to
hundreds, high sensitivity, low background. ChIRP is suitable for many lncRNAs because the design of the affinity probe
is direct in the RNA sequence and does not require knowledge of the structure or domain of the RNA. ChIRP is a novel
and rapid technique for mapping long non-coding RNA (lncRNA) genomic binding sites. The method utilizes the
specificity of the antisense spliced oligonucleotides to allow for the counting of genomic sites that bind to lncRNA [110].
RAP
RNA-centric biochemical purification is a general way to study the function and mechanism of non-coding RNA. RNA
antisense purification (RAP) is a method for selectively purifying an endogenous RNA complex from a cell extract that
allows mapping of RNA to chromatin interactions. In RAP, the user cross-links the cells to immobilize the endogenous
RNA complex and purifies these complexes by hybridization with biotinylated antisense oligonucleotides. Identification
of DNA sites interacting with the target RNA using high throughput DNA sequencing. RNA antisense purification (RAP) is
20

a method for in vivo purification of large amounts of non-coding RNA (lncRNA) complexes. RAP uses a biotinylated
antisense probe to hybridize to the target RNA to purify endogenous RNA and its associated proteins, RNA and genomic
DNA from the cross-linked cell lysates. RAP is designed to achieve explicit purification of chromatin associated with the
target lncRNA, to achieve a high-resolution map of the relevant DNA target site by sequencing the captured DNA, and to
capture any lncRNA with minimal optimization. To achieve high specificity, RAP utilizes 120 nucleotides of antisense RNA
to form a very strong hybrid with a target using denaturation conditions that disrupt non-specific RNA-protein
interactions and nonspecific hybridization with RNA or genomic DNA. To achieve high resolution, RAP uses DNase I to
digest genomic DNA to the ~ 150 bp fragment, which provides a high-resolution map of binding sites. To capture LncRNA
strongly, RAP uses multiple probe pools tiled on the entire length of the target RNA to ensure capture even in the
context of extensive protein-RNA interactions, RNA secondary structures, or partial RNA degradation [111-114].

Chromosome conformation capture
Chromosome conformation capture techniques (commonly abbreviated as 3C techniques or 3C-based methods) are a
set of molecular biological methods for analyzing the spatial organization of chromatin in cells. These methods quantify
the number of interactions between genomic loci near the 3-D space, but can be separated by many nucleotides in the
linear genome. This interaction may be caused by biological functions, such as promoter-enhancer interactions, or from
random polymer cycling, where the inadvertent physical movement of chromatin results in the locus colliding with each
other. The interactive spectrum can be analyzed directly or converted into a place for reconstructing a three-
dimensional structure.
The balance between the 3C-based methods is their range. For example, in 3C, the interaction between the two grains is
quantified. In contrast, Hi-C quantifies the interaction between all right pairs
All 3C methods begin with similar steps that are performed on the cell samples. First, the cell genome is cross-linked,
and which developers reduce the "freeze" interaction between genomic loci. Then cut the genome. Next, make a
random connection. This quantifies the proximity of the fragment, and the fragment may be connected to subsequent
fragments [115].
Subsequently, the ligated fragments are quantified using one of several techniques.
3C (one-vs-one)
Chromosome Conformation Capture (3C) was used to quantify the interaction between individual genomic loci. For
example, 3C can be used to test candidate promoter-enhancer interactions. The ligated fragments were detected using
PCR with known primers [115].
4C (one-vs-all)
The chromosome conformation capture chip (4C) captures the interaction between one locus and all other genomic
sites. It refers to a second linking step to produce a self-circulating DNA fragment for reverse PCR. Reverse PCR allows
known sequences to be used to amplify unknown sequences linked to them. Compared with 3C and 5C, the 4C
technique does not require two previous knowledge of the interacting chromosomal regions. The results derived using
4C are highly repeatable, most of the interactions detected between each other. On a single microarray, about one
million times of interaction can be analyzed [116].
5C (many-vs-many)
The chromosome conformation captures carbon-copy (5C) impedance interactions between all the limiting slices within
a given region, and its size-detail region is no greater than the mantissa. By connecting the universal primer to a partial
fragment. 5C technology overcomes the connectivity problems in the intramolecular connection step and can be used to
21

construct a harmonious interaction of a specific site. This method is not suitable for the whole genome complex
interactions, as this will require the use of millions of 5C primers [117].
Hi-C (all-vs-all)
Hi-C uses high-throughput sequencing to find the nucleotide sequence of the fragment. The original protocol uses paired
end sequencing, which retrieves short sequences from each end of each linked fragment. Thus, for a given join
fragment, the two sequences obtained should represent two different restriction fragments that are joined together in
the random connection step. The pair of sequences is aligned with the genome individually to determine the fragments
involved in the join event. Thus, all possible pairs of interactions between the segments were tested [118].

Example Workflow for a study of LncRNA and Chromosome Structure
Search for LncRNA of Interest
The transcription of LncRNA is highly cell-type specific. Therefore, LncRNAs that are highly expressed in a certain cell
type compared to other cell-type may be functional associated with the cell-specific behavior. For instance, LncRNA that
are highly transcribed in cancer cell might be involved in the activation of oncogenes or the suppression of TSGs. By
using RNA-seq, we would be able to detect the cell specific RNA expression level and thus determine LncRNA loci that is
highly expressed in a chosen cell type. Using ChIP-seq to access whether the transcription start site (TSS) of these loci is
H3K4me3 and H3K36me3, which is believed to be the epigenetic signature consists with LncRNA. To exclude the coding
potential of candidate LncRNAs, BLASTX and CSF may be helpful computational analysis tools, along with ribosomal
assays and in situ hybridization (ISH) assay to confirm the nuclear localization of candidate LncRNAs. Use double
DNA/RNA FISH to access the accumulation site of candidate LncRNA and its site of transcription. If the accumulation site
of candidate LncRNA is at or near its site of transcription, It would be likely that the candidate LncRNA is function in cis,
regulating local gene expression or chromatin organization. If the candidate LncRNA is spread across different
chromosome, then it would be act in trans.
Validate the function of LncRNA of interest
Perform siRNA knockdowns to see its impact on the behavior of cells. For example, knockdown LncRNA highly expressed
in aggressive cancer cells to see whether it could impair the invasion and proliferation of cancer cells. Test whether
overexpress of siRNA resistant LncRNA of interest could rescue the effect. If the LncRNA is act in cis, the overexpress
should be perform by targeted genome-editing technologies such as transcription activator-like effector nucleases
(TALENs) to achieve overexpression in cis. Because it is reported that nuclear-retained lncRNAs, when expressed from
transfected vectors, did not localize to the sites of their genomic counterpart regions or exert their roles in cis.
Find genes regulated by LncRNA of Interest
Apply RNA-Seq to the knockdown and overexpress model to reveal upregulate and down regulate genes. If LncRNA is
act in cis, associated genes should be located on the same chromosome near the transcription site of LncRNA of interest.
If LncRNA is act in trans, associated genes may spread across chromosomes. Use Gene Set Enrichment Analysis (GSEA) to
search for enrichment across the Molecular Signatures Database, looking for regulatory factors that may function
parallel or opposite of LncRNA of interest.
Look for interactions between genes/protein and LncRNA of interest
Apply 3C technique to measure the interaction between associated genes and the site of transcription of the LncRNAs. If
the transcription site of LncRNA is spatial proximal to the regulated genes, it indicates that chromosome looping might
be formed between.
22

Use RIP to measure the interaction between LncRNA and proteins, thus predict the possible mechanism of chromosome
structure modification of the LncRNA of interest. If LncRNA is involved in the gene regulation through mediating
chromosome looping, it may be able to find interactions between LncRNA and the chromosome looping key factors
CTCF, Mediator complex, and Cohesin. If LncRNA is involved in the gene regulation through nucleosome remodeler
recruitment, it might be able to find interactions beteween LncRNA and nucleosome remodeler.
Further applying ChIP-seq to the knockdown and overexpression model to see if the binding of chromosome looping key
factor or nucleosome remodeler to the target genes is affect by the knockdown and overexpression of the LncRNA.

23

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Abstract (if available)

Abstract Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via DNA methylation and posttranslational histone modifications is a well-documented function of lncRNA in plants, fungi, and animals. In this review, we will discuss how LncRNA can also control genome activity by affecting chromosome structure, including DNA looping and nucleosome positioning, with examples across eukaryotic kingdom, and will introduce the history and methods of LncRNA research. We explain the mechanisms of lncRNA-controlled chromatin remodeling and the implications of the functional interplay between noncoding transcription and several different chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin modifications and structure.

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Asset Metadata

Creator Peng, Zhenyu (author)

Core Title Review of long noncoding RNAs and chromosome structure

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 06/20/2017

Defense Date 05/31/2017

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chromosome structure,DNA looping,lncRNA,nucleosome positioning,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lu, Wange (committee chair), Kalra, Vijay Kumar (committee member), Tokes, Zoltan (committee member)

Creator Email pengzhenyu@yahoo.com,pengzhenyu93@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-387841

Unique identifier UC11259133

Identifier etd-PengZhenyu-5440.pdf (filename),usctheses-c40-387841 (legacy record id)

Legacy Identifier etd-PengZhenyu-5440.pdf

Dmrecord 387841

Document Type Thesis

Rights Peng, Zhenyu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA