Download Long non-coding RNAs that regulate development – Tales from the

Long non-coding RNAs that regulate
development –
Tales from the dark matter of the genome.
Professor Harvey Lodish
Whitehead Institute for Biomedical Research Departments of Biology and Biological Engineering, MIT
As metazoans evolved the number of encoded proteins
remained roughly constant whilst the genome size
exploded
Genome size
bp (millions)
Number of encoded proteins
Number of chromosomes
Drosophila
melanogaster
168
13,781 4
C elegans
100
20,424 6
Zebrafish
1505
19,929 25
Xenopus tropicalis
1700
~21,000 10
Chicken
Mouse
1050
3420
14,923 22,085 39
20
Homo sapiens
3279
21,077 23
Arabidopsis
134
27,416 5
Non-coding transcripts constitute a large
fraction of the mammalian transcriptome
Protein Coding
Transcripts
Non-coding
Transcripts
The ENCODE Project Consortium, 2007
The composition of non-coding transcripts in
the mammalian transcriptome
rRNA, snRNAs
miRNAs
snoRNAs
tRNAs
Putative
LncRNAs
Huttenhofer et al, 2005
Like mRNAs, many LncRNAs are transcribed by RNA
Polymerase II, capped, polyadenylated, and spliced. But many remain in the nucleus.
LncRNAs fall into multiple subclasses
LncRNAs fall into multiple subclasses
LncRNAs are involved in several
important biological processes
X chromosome inactivation: Xist
Epigenetic modification: HOTAIR
Genomic imprinting: H19, Air
Enhancers for neighboring genes: ncRNA-7a
p53 signaling pathway: lincRNA-p21
To be discovered
X chromosome inactivation
Epigenetic Regulation by Long Noncoding RNAs
Jeannie T. Lee Science 14 December 2012: 1435-1439
Overview of eukaryotic transcriptional control
One way of inactivating large segments of DNA by
forming “closed” heterochromatin:
methylation of cytosine residues in CG sequences;
Methylation is maintained during DNA replication
A second way of inactivating large segments of DNA by forming “closed”
heterochromatin:
Methylation of histone 3 lysine residue 9: Histone H3 lysine 9 methylation is maintained during chromosome replication.
X chromosome inactivation
LncRNAs are thought to scaffold protein
complexes and can regulate gene expression in
multiple ways
2
1
3
microRNA sponge
RNA decoy
RNP component
lncRNA
lncRNA
lncRNA
1
2
3
4
mRNA
1
2
3
4
5
6
4
3
2
lncRNA
lncRNA
mRNA
4
Recruitment of
chromatin modifiers
5
Translation
inhibition
6
Splicing
modulation
7
Degradation
1
LncRNAs are thought to scaffold protein
complexes and can regulate gene expression in
multiple ways
2
1
3
microRNA sponge
RNA decoy
RNP component
lncRNA
lncRNA
lncRNA
1
2
3
4
mRNA
1
2
3
4
5
6
4
3
2
lncRNA
lncRNA
mRNA
4
Recruitment of
chromatin modifiers
5
Translation
inhibition
6
Splicing
modulation
7
Degradation
1
Some general conclusions about
LncRNAs
• Most are expressed in very few copies per cell –
generally ~2 - ~100
• Many but not all LncRNAs are enriched in the nucleus
• Many are highly induced or repressed during specific
developmental stages and are regulated by lineageimportant transcription factors.
• Many are essential for differentiation or function of
specific cell types. LncRNAs regulate many steps in hematopoietic differentiation
LT-HSC
H19
Pluripotent
stem cells
ST-HSC
MPP
Multipotent
progenitors
CMP
CLP
GMP
MEP
HOTAIRM1
Committed
precursors
lincRNA-EPS
DLEU2
elncRNA-EC1,3
lincRNA-EC2,4,5,8,9
alncRNA-EC1,2,3,7
NeST
EGO
lincRNA-Cox2 DLEU2
LincR-Ccr2-5’AS
lincRNA
-Cox2
Mature cells
Mast cell
RBC
Megakaryocyte
Platelets
Eosinophil Neutrophil
Monocyte/
Macrophage
B cell
T cell
Dendritic
cell
NK cell
Erythropoietin is the singular hormone that prevents
apoptosis of CFU-E progenitors, allowing their terminal
proliferation and differentiation
Resolution of E 14.5 fetal liver erythroid
progenitors and erythroblasts
Scale bar: 20 µm.
In vitro culture of purified murine fetal liver CFU-E
progenitors recapitulates normal terminal erythroid
proliferation, differentiation, and enucleation
Purified TER119-negative Day 14 fetal
liver cells were cultured in vitro for one
day on fibronectin-coated plates in
medium containing serum and EPO.
EPO was removed at the end of one day.
The differentiation profiles of cultured
cells were examined after 1 and 2 days
by flow cytometry and BenzidineGiemsa staining.
After two days in culture, the number of
cells increased 20- fold and progenitors/
early erythroblasts (R1 and R2 cells)
differentiated into hemoglobinized R3 R5 cells. The differentiation profile displayed by
flow cytometry analysis correlated well
with that shown by Benzidine-Giemsa
stain. Scale bar: 20 µm.
Deep sequencing to identify erythroid- specific lncRNAs
Progenitors
Erythroblasts
Mouse E14 fetal liver:
FILTERING
ASSEMBLY
BFU-E
CFU-E
Ter119+
Poly(A)+ RNA
Total RNA
RNA-seq
ssRNA-seq
Mapping to genome (TopHat)
De novo transcript assembly
(Cufflinks)
Reliable transcript model
filters
(> 0.1 max coverage; > 200bp; >1 exon)
Coding potential filters
(pCSF<100; no PFAM domain;
no BLASTx hit; CPC<0)
Known coding transcript filter
(no sense overlap with known mRNA exons)
802 lncRNAs (657 loci)
Juan Alvarez and
Wenqian Hu
Not all fetal liver lncRNAs have already been
annotated – many are erythroid specific
ENSEMBL, UCSC &
Refseq
lncRNAs
Erythroid
lncRNAs
1648
608
194
A LncRNA with anti-apoptotic activity during the terminal differentiation of erythroid cells
ESlncRNA is highly specific to the terminal differentiating erythroid cells
ESlncRNA is a bona fide nuclear non-coding transcript
ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis
Ectopic expression of ESlncRNA protects erythroid progenitors from apoptosis caused by Epo starvation
ESlncRNA modulates apoptosis in part through repressing Pycard
Single molecule fluorescence in situ hybridization (FISH) shows
that ESlncRNA is localized to the erythroblast nucleus
Juan Alvarez
Relative ESlncRNA level
ESlncRNA is highly specific to Ter- 119 positive
terminally differentiating erythroid cells
ESlncRNA is dramatically induced during terminal
erythroid differentiation, at the same stage as ~400
erythroid important genes
Primary structure of ESlncRNA
as determined by 5’ and 3’ RACE
In erythroid cells the ESlncRNA gene contains “active”
chromatin modifications and the promoter is occupied by the
erythroid- important transcription factors Scl/Tal and GATA1
Experimental approaches for loss-offunction studies
Total Fetal Liver Cells
Lineage Depletion
(Ter119, CD3, B220, CD11b, Gr-1)
Lin- Cells
Retrovirus Infection (shRNAs)
Transduced Cells
1 Day Culture in Maintenance Medium
Switch to Differentiation Medium (+ Epo)
Molecular & Phenotypic Assays
ESlncRNA knock-down results in
inhibition of terminal erythroid
proliferation
ESlncRNA knock-down results in cell death
Conclusions
ESlncRNA is highly specific to terminal differentiating erythroid cells
ESlncRNA is a bona fide nuclear non-coding transcript
ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis
Experimental approach for gain-of-function
studies
Total Fetal Liver Cells
Lineage Depletion
(Ter119, CD3, B220, CD11b, Gr-1)
Lin- Cells
Retrovirus Infection
(Ectopic Expression of ESlncRNA)
Transduced Cells
Culture in the absence of Epo
Cell Death Analysis by Flow Cytometry
Ectopic expression of ESlncRNA in erythroid
progenitor cells is at a physiological level
Ectopic expression of ESlncRNA prevents
apoptosis caused by Epo-starvation
Ectopic expression of ESlncRNA prevents
apoptosis caused by Epo-starvation
Vector
ESlncRNA
Ectopic expression of ESlncRNA results in
repression of many apoptotic genes including
Pycard
Erythroid-specific ESlncRNA regulates
apoptosis in erythropoiesis
ESlncRNA is highly specific to the terminal differentiating erythroid cells
ESlncRNA is a bona fide nuclear non-coding transcript
ESlncRNA is required for the terminal differentiation of erythroid cells by preventing apoptosis
Ectopic expression of ESlncRNA protects erythroid progenitors from apoptosis caused by Epo starvation
ESlncRNA modulates apoptosis in part through repressing Pycard
Fetal liver lincRNAs exhibit an extremely high
degree of cell and tissue specificity
Fetal liver lincRNAs exhibit an extremely high
degree of cell and tissue specificity
Dynamic expression of distinct lncRNA
subclasses during erythropoiesis
Selection of 12 new candidate erythroid lncRNAs:
!"#$%$"&'()#*+,-.(/01(23#*&%0#")(!4"1"*&'1%5"&%0#(
• Induced during terminal erythroid differentiation
• Promoter bound by key erythroid transcription factors
• Conserved in placental mammals
()*+,--./0#
1,23456./0#
7/0-,+856./0#
$%&'#
!"#
%459,0654#:5::54-#
!"#$%$"&'()#*+,-.(/01(23#*&%0#")(!4"1"*&'1%5"&%0#(
Selection of candidate lncRNAs:
• Abundant in and specific for erythroid cells
!"##$%&'(%)"*")"+,&
In situ single molecule RNA FISH: Most candidate
lncRNAs are enriched in the nucleus
Knockdown of most candidate lncRNA does not block
glycophorin (Ter119) induction
But knockdown of most candidate lncRNA blocks
later red cell development, as measured by
enucleation
Conclusion: Multiple Types of Long Non-coding
RNAs Regulate Red Blood Cell Development
• We combined genome-wide surveys of expression,
chromatin state and transcription factor
occupancy in differentiating primary mouse fetal
liver erythroid cells to systematically characterize
each lncRNA subclass. • Binding of the key erythroid transcription factors
GATA1 and TAL1 at lncRNA and mRNA
promoters is typically accompanied by gain of
H3K4me2 along with transcriptional activation. • We focused on lncRNAs from each subclass that
are abundantly upregulated during terminal
differentiation, including novel erythroid-specific
lncRNAs conserved in humans and that are
predominantly nuclear. Loss-of-function studies
revealed that 12 of these lncRNAs are necessary
for the proper maturation of enucleated
erythroblasts. • Together, these results indicate that lncRNAs of
diverse genomic origins are important
components of the regulatory networks
underlying development of the erythroid lineage. Pipeline for de novo
lncRNA discovery
in adipocytes
BAT = brown adipose tissue
iWAT = inguinal fat tissue
eWAT = epididymal fat tissue
Almost all of the sequenced lncRNAs are expressed in a
highly cell- and tissue- specific fashion
About half of the sequenced mRNAs are expressed in a
highly cell- and tissue- specific fashion
About half of the sequenced mRNAs are expressed in a
highly cell- and tissue- specific fashion
Wenqian Hu and Juan Alvarez-Dominguez
A proud 31 year tradition continues: July 27, 2013