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Long Noncoding RNA Mediates Both Activation and Repression

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立群 謝

on 5 October 2016

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Transcript of Long Noncoding RNA Mediates Both Activation and Repression

Pam3CSK4 induced the transcription of numerous protein-coding genes involved in the immune response , as well as 62 lncRNAs.
The most significantly induced lncRNAs tended to occur in chromosomal regions where also higher expression of immune genes coregulated.
lincRNA-Cox2
was among the most highly induced lncRNAs and proximal to the prostaglandin-endoperoxide synthase 2 [Ptgs2(Cox2)] gene
Long Noncoding RNA Mediates Both Activation and Repression of Immune Response Genes
Science 16 August 2013:
Vol. 341 no. 6147 pp. 789-792
Susan Carpenter et al.

Presenter: Kevin, LI-Chun Hsieh / Supervisor: Hsueh-Fen Juan

Examined the
protein-coding capacity
of lincRNA-Cox2 by assessing its association with polysomes (ribosome complex) within cells.
BMDMs were treated with :
Cycloheximide: trap ribosomes on RNA molecules
Harringtonine - disrupting translation.
EDTA - disrupting all RNA-protein interactions
Cell lysates were
fractionated
and
RNA analyzed
by qPCR.
Next, generated BMDM cell lines in which lincRNA-Cox2 expression was silenced by shRNA

To identify potential targets (signal) of lincRNA-Cox2, RNA-seq in both control and lincRNA-Cox2–silenced cells before and after stimulation with Pam3CSK4 was done.

Generating macrophages that ectopically expressed lincRNA-Cox2

Macrophages that ectopically expressed lincRNA-Cox2 had
decreased
levels of Ccl5, Irf7, and other ISGs

lncRNAs can be found in the nucleus, cytoplasm, or both.

We examined the localization of lincRNA-Cox2 in macrophages by qRT-PCR on RNA isolated from nuclear or cytosolic fractions

The innate immune system coordinates host defenses through pattern recognition receptors [Toll-like receptors (TLRs)], which recognize microbial products and induce the expression of hundreds of proteins involved in antimicrobial defense and adaptive immunity
Introduction - Immune System
Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes.
lncRNAs are operationally defined as RNA genes larger than
200
bp that do
not
appear to have coding potential.
Introduction - Long non-coding RNAs
To find out lncRNAs:

DNA microarray
Chromatin Marks
RNA - seq (NGS)
Function of lncRNA
Embryonic stem cell pluriopotency
Cell Cycle regulation
Disease development - Cancer, Alpha-Thalassaemia, Alzheimer’s Disease, Myotonic Dystrophies
Ribosomal profiling

Codon substitution frequency analysis
Gain or loss of function test
Drive the formation of ribonucleic protein
complexes, influencing the regulation of gene expression.
Single, membrane-spanning, non-catalytic receptors expressed in sentinel cells such as macrophages that recognize molecules derived from microbes.
Once microbes have breached physical barriers such as the skin /GI mucosa, they are recognized by TLRs, then activate proteins involved in antimicrobial defense and adaptive immunity.
TLRs with the IL-1 receptors form "Interleukin-1 Receptor / Toll-Like Receptor Superfamily"; TLR 1~13 subtypes.
Toll-like Receptor
After lipopolysaccharide (LPS) stimulation, lncRNAs are differentially regulated in virus-infected cells and in
dendritic cells
.
lncRNA NeST was shown to control susceptibility to Theiler’s virus and Salmonella infection in mice through epigenetic regulation of the interferon-g (IFN-g) locus.
Although
lncRNAs
can be induced in
innate immune
cells, whether lncRNAs act as regulators of gene expression in
innate immunity
is unknown.
Model: mouse bone marrow–derived macrophages (BMDMs) stimulated with the synthetic bacterial lipopeptide Pam3CSK4 - a Tlr2 ligand.

Analysis method: whole-transcriptome analysis (RNA-seq)
The inner track shows log2 relative change for protein- coding genes: red - immune genes; blue-other genes. The outer track shows log2 relative change for all lncRNAs.
Circos. Genome Res. 2009. 19: 1639-1645
A previous study demonstrated that lncRNA- Cox2 was induced in
dendritic cells
after stimulation with LPS.
Whether lincRNA-Cox2 regulates the inflammatory response that is associated with
TLR
signaling is
unknown
.
qPCR in bone marrow–derived dendritic cells (BMDCs)
Induction of lincRNA-Cox2 and its neighboring gene
Ptgs2 (Cox2) was dependent on the Tlr adaptor protein
MyD88 and transcription factor NF-kB signaling.
Cox-2 is regulated by Toll-like receptor signaling in
Prostaglandins (PGs) production.
Comparison between:

1. Gapdh mRNA
2. lincRNA-Cox2
3. lncRNA-Eps, was proved to be noncoding
Kozak strength analysis:
Strength of translation initiation signal sequence
Summary:
lincRNA-Cox2 is unlikely to encode a protein product.
- - + +
upregulated
down regulated immune genes
Chemokines (
Ccl5
, Cx3cl1), Chemokine receptors (Ccrl),
IFN-stimulated genes (
Irf7
, Oas1a, Oas1l, Oas2,
Ifi204
, and Isg15)
Tlr1,
IL6
(interleukin-6), IL23a..
GO enrichment analysis
RNA profiling technology (nCounter; Nanostring).
Increase in expression of
Irf7
,
Ccl5 (Rantes)
and ISGs
Pam3CSK4-induced expression of Tlr1 and
IL6
decreased
ELISA data of Ccl5 (up), Il6 (down)
To distinguish between
IFN-dependent
and
IFN-independent
targets of lincRNA-Cox2, they
silenced lincRNA-Cox2
in macrophages lacking the type I IFN a/b receptor (IFN a/bR KO).
Expression levels of lincRNA-Cox2 (F),
Ccl5 (G), Irf7 (H), and Ifi204 (I)
were measured by qRT-PCR.
Regulation of Irf7 and Ifi204 appeared to be
dependent to type I IFN
IFN-stimulated genes (ISGs), increased by
linc shRNA
before KO
Ccl5 was regulated by lincRNA-Cox2
independently
of type I IFN
These data indicate that lincRNA-Cox2 regulates distinct classes of
immune genes both
basally
and
after TLR stimulation (IFN dependent)
.
lincRNA-Cox2
represses Ccl5
while simultaneously
enhancing
the expression
of
Il6
(TLR-induced one).
To identify binding partners for lincRNA-Cox2, we incubated in vitro–transcribed biotinylated lincRNA-Cox2 as well as an antisense lincRNA-Cox2 control RNA with nuclear or cytosolic extracts of macrophages.
www.activemotif.com
Differentially expressed bands were excised and RNA binding proteins
hnRNP-A/B
and
hnRNP-A2/B1
identified by
Mass Spectrometry
.
The ability of hnRNP-A/B and hnRNP-A2/B1 to bind
lincRNA-Cox2 was confirmed.
hnRNPs also act as mediators of lncRNA-induced transcriptional repression.
hnRNP-A/B has been linked to transcriptional repression of some genes and hnRNP-A2/B1 and hnRNP-A/B have been shown to associate.

They hypothesized that lincRNA-Cox2 regulates the transcription of aforementioned immune genes
by associating with these hnRNPs.
They knock down hnRNP-A/B or hnRNP-A2/B1 by shRNA in macrophages.
Knockdown of hnRNP-A/B and hnRNP-A2/B1
did not modulate lincRNA-Cox2 level but
increase Ccl5 protein levels
in both unstimulated and Pam3CSK4-stimulated cells.
Next, they examined the occupancy of RNA polymerase II at the promoters of the Ccl5 and Irf7 genes using chromatin immunoprecipitation (ChIP: RNAPol-ChIP qPCR):

Increased
levels of RNA polymerase II (Pol II) at the Ccl5 and Irf7 promoters when
lincRNA-Cox2 or hnRNPs were silenced
in unstimulated cells
Endogenous Control
Silencing of hnRNP-A2/B1 or
hnRNP-A/B in cells overexpressed
lincRNA-Cox2 reversed the inhibitory
effect of lincRNA-Cox2 on Ccl5
expression
Summary: confirm hnRNP-A/B and hnRNP-A2/B1 form a complex with lincRNA- Cox2 to repress the transcription of immune genes.
They propose a model - TLR signaling induces lincRNA-Cox2, which serve as repressors and activators of genes through interactions with various regulatory complexes.

lncRNAs represent a component of the innate immune response that can both restrain and promote aspects of inflammatory signaling.

Conclusion
Thanks for Attention...
and...Smile~
Workflow for predicting lncRNAs using deep RNA sequencing.
Methods 63 (2013) 50–59.
Function Mechanism
Anatomical Classification of lncRNA
Nature 458, 223–227 (2009), mBio 1, e00206 (2010), Aune, J. Immunol. 189, 2084–2088 (2012)
Backgroud - immune system & lncRNA
Purpose
Materials and Methods
Nat. Methods 5, 621–628 (2008)
Other two upregulated lncRNA:
Nature 458, 223–227 (2009)
Structure of lincRNA-Cox2
Not discussed in this study..
Gastroenterology. 2006:862-77.
lncRNA-Cox2 / Ptgs levels in Knockout Myd88
lincRNA-Cox2 / Ptgs levels after blocking NFkB
Proximal to Ptgs gene
Discussion
Bias in "gain of function" and "loss of function" maneuver.
Silencing lincRNA-Cox2 not alter expression of Ptgs?
Better way to study signal pathway? Protein-protein interaction?

Summary
(lincRNA; i = intergenic)
Loss of Function Study
rtPCR data
rtPCR data
?
(Regulated upon Activation, Normal T cell Expressed and Secreted)
Gain-of-function studies
Many lncRNAs regulate transcription through their interactions with chromatin-modifying complexes or with heterogeneous nuclear ribonucleoproteins (hnRNPs).

To identify binding partners for lincRNA-Cox2, they incubated in vitro–transcribed
biotinylated lincRNA-Cox2
as well as an
antisense lincRNA-Cox2 control RNA
with
nuclear
or
cytosolic
extracts of macrophages and subjected RNA binding proteins to mass spectrometry for identification.
Immunoprecipitation Study
Streptavidin bead- bind to biotin
antibodies to hnRNP-A/B and hnRNP-A2/B1
Summary
Heat map of differentially regulated genes of control shRNA, hnRNP-A/B shRNA, or hnRNP-A2/B1 shRNA expressing BMDMs stimulated with Pam3CSK4
Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites
Block the overexpression function by knock down hnRNP?
lncRNA-COX2 is upregulated in mouse macrophage after stimulation with LPS and Tlr2 ligand through RNA-seq. This event happened in concurrent with PGs expression and dependent to MyD88 and NFkB.
Knock down (loss of function) of lncRNA-COX2 upregulate Ccl5 and IFN-stimulated genes (ISGs). Down-regulation of Tlr1, Il6 and Il23a. In IFN-alpha/beta receptor KO mice, the lncRNA-COX-2 and CCl-5 are independently but Irf7 and Ifi204 were dependent to IFN receptor.
lncRNA-COX2 overexpression (gain of function) upregulated IL-6 and downregulated Ccl5, Irf7, and other ISGs.
Immunoprecipitation study with LC/MS revealed the binding protein of lncRNA-COX2, hnRNP-A/B or hnRNP- A2/B1
Increased RNA pol II recruitment to CCl-5 promotor after knockdown of lncRNA-COX2, hnRNP-A/B or hnRNP- A2/B1 genes are noted by ChIP.
CCl-5 down-regulation effect by lncRNA-COX2 overexpression can be reversed by knockdown hnRNP-A/B or hnRNP- A2/B1 genes
Summary and Take home messages -1
Summary and Take home messages -2
Full transcript