coordinate homeotic gene expression
Kevin C. Wang1,2, Yul W. Yang1*, Bo Liu3*, Amartya Sanyal4, Ryan Corces-Zimmerman1, Yong Chen5, Bryan R. Lajoie4,
Angeline Protacio1, Ryan A. Flynn1, Rajnish A. Gupta1, Joanna Wysocka6, Ming Lei5, Job Dekker4, Jill A. Helms3
& Howard Y. Chang1
The genome is extensively transcribed into long intergenic non-
coding RNAs (lincRNAs), many of which are implicated in gene
silencing1,2. Potential roles of lincRNAs in gene activation are
much less understood3–5. Development and homeostasis require
coordinate regulation of neighbouring genes through a process
termed locus control6. Some locus control elements and enhancers
transcribe lincRNAs7–10, hinting at possible roles in long-range
control. In vertebrates, 39 Hox genes, encoding homeodomain
transcription factors critical for positional identity, are clustered
in four chromosomal loci; the Hox genes are expressed in nested
genomic position from 39 to 59of the cluster11. Here we identify
that coordinates the activation of several 59 HOXA genes in vivo.
Chromosomal looping brings HOTTIP into close proximity to its
target genes. HOTTIP RNA binds the adaptor protein WDR5
directly and targets WDR5/MLL complexes across HOXA, driving
proximity is necessary and sufficient for HOTTIP RNA activation
mit information from higher order chromosomal looping into
chromatin modifications, lincRNAs may organize chromatin
domains to coordinate long-range gene activation.
We examined chromosome structure and histone modifications in
human primary fibroblasts derived from several anatomic sites12, and
found distinctive differences in the HOXA locus. High throughput
its higher order structure is dependent on positional identity. In ana-
tomically distal cells (for example, foreskin and foot fibroblasts), we
of constituent Hox genes), pointing to a compact and looped con-
formation. In contrast, no long-range chromatin interactions are
detected within the transcriptionally silent 39 HOXA which seems
largely linear (Fig. 1a). Strikingly, anatomically proximal cells (for
example, lung fibroblasts) have the diametrically opposite pattern.
The ON and OFF states of Hox and other key developmental genes
are maintained by the MLL/Trithorax (Trx) and polycomb group
(PcG) proteins, which mediate trimethylation of histone H3 lysine 4
(H3K4me3) to activate genes or lysine 27 (H3K27me3) to repress
genes14. The portions of HOXA in tight physical interaction are
marked by broad domains of H3K4me3, whereas H3K27me3 marks
the physically extended and transcriptional silent regions (Fig. 1a).
are two lincRNA loci that exhibit distinct chromatin modifications.
The 39element has been previously identified as the myelopoiesis-
associated lincRNA HOTAIRM1 (ref. 15). The 59 element, for which
we suggest the name HOTTIP for ‘HOXA transcript at the distal tip’,
exhibits bivalent H3K4me3 and H3K27me3, a histone modification
pattern associated with poised regulatory sequences16. Comparison
with RNA polymerase II occupancy and RNA expression showed that
do not require HOTTIP transcription, but transcription of HOTTIP is
left). Chromatin immunoprecipitation (ChIP) analysis confirmed that
the HOTTIP gene is occupied by both polycomb repressive complex 2
(PRC2) and MLL complex, consistent with the bivalent histone marks
(Supplementary Fig. 1a).
HOTTIP transcription yields a 3,764-nucleotide, spliced and poly-
adenylated lincRNA that initiates ,330 bases upstream of HOXA13.
Only the strand antisense to HOXA genes is transcribed (Supplemen-
tary Fig. 1b). Genes near the 59 end of each HOX cluster tend to be
expressed in more posterior and/or distal anatomical locations.
Consistent with its genomic location 59 to HOXA13, HOTTIP is
expressed in distal and/or posterior anatomic sites (Fig. 1b). In situ
hybridization of developing mouseandchick embryos confirmedthat
HOTTIP is expressed in posterior and distal sites in vivo, indicating a
conserved expressionpatternfromdevelopment to adulthood(Fig. 1c
and Supplementary Fig. 1c). Even in distal cells where HOTTIP is
expressed, its RNA level is very low and estimated to be ,0.3 copies
per cell (Supplementary Fig. 2).
We employed small interfering RNAs (siRNAs) to knock down
HOTTIP RNA in fibroblasts from a distal anatomic site (foreskin),
and examined expression of 59 HOXA genes by quantitative reverse
transcription PCR. Notably, HOTTIP RNA knockdown abrogated
expression of distal HOXA genes across 40kilobases with a trend
dependent on the distance to HOTTIP. The strongest blockade was
observed for HOXA13 and HOXA11, with progressively less severe
effects on HOXA10, HOXA9 and HOXA7 (Fig.2a).Theeffect on gene
transcription appeared to be unidirectional, as there were no appre-
ciable changes in the levels of EVX1, located ,40 kilobases 59 of the
HOXA cluster (data not shown). HOTTIP knockdown did not affect
nor induce antisense transcription at its own locus (Fig. 2b, Sup-
plementary Fig. 3a). Several independent siRNAs targeting HOTTIP
yielded similar results (Supplementary Fig. 3b). These results indicate
that HOTTIP RNA is necessary to coordinate activation of 59 HOXA
We next addressed the function of HOTTIP RNA in vivo in the
developing chick limb bud (Fig. 2c). Whereas prior genetic studies
of noncoding RNAs (ncRNAs) involved deletion or insertion into
the gene locus17, we wished to distinguish the functions of HOTTIP
H3K4 and H3K27 methylation independent of transcription (Fig. 1a),
*These authors contributed equally to this work.
1Howard Hughes Medical Institute, Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA.2Department of Dermatology, University of California San
University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.6Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305.
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and the precise genomic distance between upstream enhancer ele-
ments and Hox genes is critical for their proper colinear activation17.
replication-competent retroviruses can deliver short-hairpin RNAs
(shRNAs) with high penetrance and precise spatiotemporal control18
function is highly redundant with that of the HoxD locus, which
allowed us to assess altered HoxA expression patterns without major
changes in anatomic landmarks20. We injected retroviruses carrying
knockdown samples after 2–4days. Knockdown of HOTTIP RNA by
two independent shRNAs in limb buds decreased expression of
HoxA13, HoxA11 and HoxA10—again with a graded impact depend-
ing on genomic proximity to HOTTIP gene. Vector control or an
expression (Fig. 2d). In situ hybridization on whole embryos (Fig. 2e)
and sections (Supplementary Fig. 5) revealed that HOTTIP RNA most
limb bud, where the 59 HoxA genes are most strongly expressed. By
stage 36, limbs depleted of HOTTIP RNA showed notable shortening
and bending of distal bony elements, including the radius, ulna and
third digit (,20% length reduction for each compared to contralateral
t-test, Fig. 2f). This phenotype resembled some of the defects in mice
that HOTTIP RNA controls activation of distal Hox genes in vivo.
The broad impact of HOTTIP RNA on gene activation across the
HOXA locus is reminiscent of the broad domains of chromatin modi-
fications demarcating active and silent chromosomal domains12. 5C
analysis of control and HOTTIP-depleted cells showed little change in
higher order chromosomal structure, indicating that the chromo-
somal looping is pre-configured and upstream of gene expression
HOTTIP RNA level
100 200 300 400
HOTTIP RNA Sense control
Full length 3,764 nucleotides
A4 A3 A2 A1
A11 A10 A9A7
Distal cells Proximal cells
A4 A3 A2 A1
A11 A10 A9 A7
A3 A2 A1
A11 A10 A9
Figure 1 | HOTTIP is a lincRNA transcribed in distal anatomic sites.
a, Chromatin state map of distal versus proximal cells. Top panels,
chromosome conformation capture-carbon copy (5C) analysis of distal
(foreskin) and proximal (lung) human fibroblasts. Heat map representations
in foreskin and lung fibroblasts. Red intensity of each pixel indicates relative
interaction between the two points on the genomic coordinates. The diagonal
represents frequent cis interactions between regions located in close proximity
along the linear genome. 5C signals that are away from the diagonal represent
long-range looping interactions. Bottom panels, chromatin occupancy across
HOXA. x-axisisgenomiccoordinate;y-axisdepictsoccupancyofthe indicated
histone marks or protein (ChIP/input). Box and arrows highlight chromatin
states of HOTTIP gene. b, HOTTIP RNA expression in primary human
fibroblasts from 11 anatomic sites. Means6s.d. are shown (n52). c, In situ
hybridization of HOTTIP RNA in E13.5 mouse embryo.
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(Supplementary Fig. 6a). In contrast, HOTTIP RNA knockdown led to
broad loss of H3K4me3 and H3K4me2 across the HOXA locus, most
prominently over 59 HOXA and HOTTIP gene itself (Fig. 3a, Sup-
plementary Figs 6b and 7). HOTTIP RNA knockdown also increased
H3K27me3 focally over HOTTIP gene, but had little impact on
H3K27me3 across HOXA. These results indicate that HOTTIP RNA
is required for maintenance of H3K4me3 across the HOXA. These
findings also imply that loss of 59 HOXA gene transcription upon
HOTTIP RNA knockdown is likely to be due to loss of H3K4me3
(or other changes) rather than ectopic spread of H3K27me3.
H3K4methylationofthe HOX lociiscarried out bythe MLLfamily
ofcomplexes24. Inmammals,atleast sixMLL familymembers ofSET-
plex of WDR5, ASH2L, RBBP5, as well as with other proteins, for
substrate recognition and genomic targeting24. Genetic analyses indi-
cate that MLL1 and 2 are most essential for HOX gene expression in
fibroblasts25, and MLL1inparticularisrecruited topromoters ofHOX
genes to maintain their activation states26. In distally-derived human
fibroblasts,MLL1and WDR5densely occupiedextendedregionofthe
59 HOXA cluster, coincident with the H3K4me3domain, with specific
‘peaks’ of occupancy near the transcriptional start sites (TSS) of mul-
tiple 59 HOXA genes (Fig. 3b). Strikingly, HOTTIP RNA knockdown
in diffuse and less intense binding of MLL1 and WDR5 across HOXA
cluster, most prominently over the 59 HOXA domain. HOTTIP RNA
critical for maintaining a specific pattern of MLL complex occupancy
or more subunits of the MLL complex. Purified, in-vitro-transcribed,
full-length HOTTIP RNA bound specifically to recombinant
glutathione-S-transferase-conjugated WDR5 (GST–WDR5), but not
TERF1; Fig. 4a, b). The C terminus of MLL1, containing the SET
domain, bound non-specifically to all RNAs, consistent with previous
studies27. Immunoprecipitation of endogenous WDR5 from two dif-
ferent cell lines each specifically retrieved endogenous HOTTIP RNA
(Fig. 4c), indicating that WDR5 and HOTTIP RNA interact in living
HOTTIP A13 A11 A10 A9 A7
RNA (fold by qRT–PCR)
1.0 0.40.4 1.3
0.6 0.7 1.0
Injection stage 13Harvest stage 24
RT–PCR/in situ hybridization
Harvest stage 30
RNA fold (siHOTTIP/siGFP)
HOXD13 HOXD11 HOXD10 BID
Figure 2 | HOTTIPisrequiredforcoordinateactivationof59HOXAgenes.
a, b, Knockdown of HOTTIP RNA abrogates expression of 59 HOXA genes in
foreskin fibroblasts (a), but not HOXD or BID genes (b). Means1s.d. are
shown (n53). GFP, green fluorescent protein. c, Schematic of chick RNAi
experiment. d, HOTTIP RNA is required for 59 HoxA gene expression in vivo.
RT–PCR of the indicated genes from control or HOTTIP-depleted distal limb
each band. GAG signal confirms successful retroviral transduction in all cases.
e, In situ hybridization of 59 HoxA genes in chick limb buds. Arrowheads
highlight distal domains of high HoxA gene expression that are affected by
HOTTIP knockdown. f, Shortening of distal bony elements in HOTTIP-
depleted forelimbs. Alcian blue staining highlights the skeletal elements. Red
and purple lines highlight radius and 3rd digit lengths, respectively.
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cells. Immunoprecipitation of an epitope-tagged WDR5 from a stable
celllinethat previouslyenabled stoichiometricpurificationofWDR5-
interacting proteins28also specifically retrieved HOTTIP RNA
(Supplementary Fig. 9). Knockdown of WDR5 broadly inhibited
expression of 59 HOXA genes, and also abrogated HOTTIP transcrip-
tion, demonstrating mutual interdependence between HOTTIP RNA
and WDR5 (Fig. 4d).
HOTTIP RNA seems to regulate genes in cis, due to its low copy
number, distance dependence of HOXA target gene activation on
endogenous HOTTIP, and the physical proximity of HOTTIP and
its target genes as seen in 5C. Indeed, ectopic expression of HOTTIP
RNA by retroviral transduction of lung fibroblasts, which do not
express HOTTIP, failed to activate expression of distal HOXA genes,
and didnot change H3K4me3 and H3K27me3 patterns acrossHOXA
endogenous HOTTIP, ectopic HOTTIP expression did not induce 59
HOXA genes, nor rescue the effects of depleting endogenous nascent
HOTTIP RNA (Supplementary Fig. 11). The lack of response in fore-
in these cells, indicating that the protein partners of HOTTIP are all
present and target genes are receptive. Ectopically expressed HOTTIP
RNA, being transcribed from retroviral insertion sites scattered
randomly in the genome, may not be able to find 59HOXA genes. In
contrast, endogenous HOTTIP RNA is directly positioned near the
59 HOXA genes by chromosomal looping, allowing interaction and
To test the requirement of an exogenous targeting mechanism, we
engineered an allele of HOTTIP RNA that can be artificially recruited
to a reporter gene. Addition of five copies of the BoxB RNA element29
Recruitment of HOTTIP RNA to a silent GAL4 promoter is not suf-
ficient to initiate transcription, but can significantly boost transcrip-
tion if the promoter is also bound by WDR5 and transcriptionally
active (Fig. 4e). By uncoupling the sites of HOTTIP transcription
versus HOTTIP RNA function, this experiment indicates that the
proximity of HOTTIP RNA—rather than the act of transcription—
maintains target gene expression. To further support the functionality
ofHOTTIP RNA,deletionanalysis identified a ,1kb domaininthe59
A11 A10A9A7 A6 A5 A13
A11 A10A9 A7 A6 A5 A13
Figure 3 | HOTTIP RNA is required for the active chromatin state of 59
HOXA cluster. a, Knockdown of HOTTIP RNA broadly decreases H3K4me3
across 59 HOXA locus but focally affects H3K27me3 at HOTTIP gene. Display
is as in Fig. 1a. b, Knockdown of HOTTIP RNA abrogates peaks of MLL1 and
WDR5 occupancy near TSSs of 59 HOXA genes and leads to accumulation of
these proteins at HOTTIP gene itself. Arrows highlight peaks of MLL1 and
WDR5 occupancy; open arrowheads highlight chromatin state of HOTTIP
gene upon HOTTIP RNA knockdown.
(fold by qRT–PCR)
RNA IP (fold by qRT–PCR)
RNA (fold by qRT–PCR)
WDR5 HOTTIP A13A11A10A7
Figure 4 | HOTTIP RNA programs active chromatin via WDR5.
a, Summary of RNA–protein interaction studies. Each of the indicated
recombinant protein was purified and used to retrieve purified HOTTIP RNA
or control histone RNA in vitro. Only GST–WDR5 specifically retrieved
HOTTIP. b, HOTTIP RNA binds directly and specifically to WDR5. Left,
purified GST and GST–WDR5 are visualized by SDS–PAGE and Coomassie
Blue staining. Right, retrieved RNAs are quantified by qRT–PCR. c, HOTTIP
RNA binds specifically to WDR5 in cells. Immunoprecipitation (IP) of
endogenous WDR5 protein from PC3(prostate)andT24(bladder)carcinoma
cells specifically retrieved HOTTIP, but not control IPs with IgG or chromatin
binder SIRT6. U1 spliceosomal RNA served as negative control. d, WDR5 is
required for 59 HOXA gene expression, including HOTTIP RNA. e, HOTTIP
RNA recruitment potentiates transcription. Left, the BoxB tethering system.
BoxB–RNA specifically binds lN fused to GAL4 DNA binding domain,
recruiting the complex to a UAS-luciferase reporter gene. After transient
luciferase activity after co-transfection of the indicated constructs. *P,0.05
are shown for all panels.
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ently dominant negative manner (Supplementary Fig. 12b).
a switch to produce HOTTIP lincRNA, which binds to and targets
WDR5–MLL complexes to the 59 HOXA locus, yielding a broad
domain of H3K4me3 and transcription activation (Supplementary
Fig. 13). The mutual interdependence between HOTTIP RNA and
WDR5 creates a positive feedback loop that maintains the ON state
of the locus. These findings provide an integrated view linking three
dimensional genome organization to dynamic programming of chro-
matin states, and ultimately to developmental pattern formation.
H3K4 methylation is a feature of almost all transcribed genes, and
MLL family proteins are involved in many cell fate decisions in
development and disease24. Our findings suggest that additional
activities8–10, may also be involved in gene activation by programming
active chromatin states, and highlight WDR5 and other WD40 repeat
proteins as candidate adaptors that link chromatin remodelling com-
plexes to lincRNAs. Cis-restricted lincRNAs may be ideally suited to
link chromosome structure and gene expression. Because such
modification via RNA recruitment of enzymatic activities, and thus
into gene expression.
High throughput chromosome confirmation capture (5C) was performed on
foreskin and lung fibroblasts, as well as foreskin fibroblasts treated with control
or siRNA against HOTTIP RNA, as described13. siRNA knockdown experiments
on cultured human fibroblasts and qPCR were performed as described previ-
ously12. ChIP-chip was performed as described12using ultra-high-density HOX
tiling arrays. Full-length HOTTIP RNA was cloned by 59 and 39 rapid amplifica-
tion of cloned/cDNA ends (RACE). Single-molecule RNA-fluorescent in situ
hybridization (FISH) was performed using a pool of fluorescently-labelled oligo-
nucleotides specific to HOTTIP RNA. In vivo HOTTIP RNA knockdown in chick
for RNA in situ hybridization, immunohistochemistry and whole-mount limb
analysis, respectively. RNA-immunoprecipitation with WDR5 was performed as
described12.Tethering experiments were done in 293T cells with co-transfections
of various constructs containing a upstream activating sequence (UAS)-luciferase
reporter, GAL4-WDR5, BoxB alone, and BoxB fused to full-length HOTTIP or
full-lengthLacZ; cellswere lysed 48h after transfection andluciferase activitywas
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 23 February 2010; accepted 12 January 2011.
Published online 20 March 2011.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank C. Tabin for chick Hox gene probes, M. Scott and
members of our labs for input, and M. Lin for use of the confocal microscope and
imaging expertise. Supported by grants from the California Institute for Regenerative
Medicine (H.Y.C., J.W.), the National Institutes of Health (HG003143 to J.D.), and the
Scleroderma Research Foundation (H.Y.C.). K.C.W. is a recipient of a Dermatology
Distinguished Young Scholar Award. H.Y.C. and M.L. are Early Career Scientists of the
Howard Hughes Medical Institute.
Author Contributions K.C.W., R.A.G. and H.Y.C. initiated the project; K.C.W. and H.Y.C.
designed the experiments; K.C.W., Y.W.Y., B.L., A.S., R.C.-Z., B.R.L., A.P., R.A.F., J.D. and
J.A.H. conducted the experiments and analysed the data; Y.C. and M.L. purified the
recombinant proteins; J.W. provided antibodies and cell lines; K.C.W. and H.Y.C.
prepared the manuscript with inputs from all co-authors.
Author Information Sequence for human HOTTIP RNA has been deposited with
GenBank under the accession number GU724873. Microarray data are deposited in
Gene Expression Omnibus (GEO) under accession number GSE26540. Reprints and
permissionsinformationisavailable atwww.nature.com/reprints.The authors declare
no competing financial interests. Readers are welcome to comment on the online
version of this article at www.nature.com/nature. Correspondence and requests for
materials should be addressed to H.Y.C. (email@example.com).
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Cells. Primary human fibroblasts derived from different anatomic sites were as
which have been confirmed in vivo33,34,37.
Chromatin immunoprecipitation followed by microarray analysis. ChIP-chip
was performed using anti-H3K27me3 (Abcam), anti-H3K4me3 (Abcam), anti-
H3K4me2 (Abcam), anti-histone H3 (Abcam), anti-PolII (Abcam), anti-MLL1
(gift of R. Roeder), and anti-WDR528antibodies as previously described12.
Chromatin from each indicated cell type or RNAi treatment is split into multiple
input chromatin were competitively hybridized to custom tiling arrays interrog-
ating human HOX loci at 5-bp resolution as previously described12.
restriction sites using 5C primer design tools previously developed13and made
available online at http://my5C.umassmed.edu (ref. 30). Reverse primers were
designed for fragments overlapping a known transcription start site from
GENCODEtranscripts38, oroverlapping a startsite asexperimentallydetermined
tive sequences prevented the design of a sufficiently unique 5C primer. Primers
settings were: U-BLAST: 3; S-BLAST: 130: 15-MER: 1320; MIN_FSIZE: 40;
MAX_FSIZE: 50000; OPT_TM: 65; OPT_PSIZE: 40. DNA sequence of the uni-
versal tails of forward primers was CCTCTCTATGGGCAGTCGGTGAT; DNA
sequence for the universal tails of reverse primers was AGAGAATGAGGAACC
CGGGGCAG. A 6-base barcode was included between the specific part of the
primers and the universal tail. In total 17 reverse primers and 90 forward primers
were designed in the 500kbHoxA1locus(ENm010) andhencea total of1,530 cis
interaction were interrogated in this region. Primer sequences are available sepa-
rately (Supplementary Table 1).
3C was performed with HindIII as previously described40separately for fetal
lung and foreskin fibroblasts (FB) and also for the control and HOTTIP knock-
of HoxA1 region were mixed with either the ENCODE random region (ENr)
primer pool comprising of 2,673 forward and 523 reverse primers (covering 30
additional ENCODE regions) or the ENr313 primer pool comprising of 57 for-
ligation products were amplified using a pair of universal primers that recognize
the common tails of the 5C forward and reverse primers described above and
pooled together. To facilitate paired-end DNA sequence analysis on the Illumina
GA2 platform, paired-end adaptor oligonucleotides were ligated to the 5C library
using the Illumina PE protocol and PCR amplification of the library was carried
fetal lung FBs we obtained 7,625,276 and 10,947,424 mapped reads for two bio-
logical replicates of which 1,339,861 and 242,301 could be specifically mapped
back to interactions within ENm010 using Novoalign (http://www.novocraft.
com), respectively. For two biological replicates of foreskin FBs we obtained
7,311,386 and 5,731,107 mapped reads of which 2,752,789 and 66,769 could be
mapped back to the ENm010 region, respectively. In the case of the knockdown
yielded 4,909,482 mapped reads whereas HOTTIP knockdown foreskin FB had
5,565,389 mapped reads of which 39,168 and 38,950 could be mapped back to
ENm010 for control GFP and HOTTIP knockdown, respectively. In the set with
fetal lung and foreskin fibroblast samples, 5C for ENm010 was multiplexed for
deep sequencing with 5C of one other region, ENr313; in the set containing the
knockdown samples, ENm010 was multiplexed with 5C of 30 other genomic
regions. The different extent of multiplexing resulted in different number of
sequencing reads mapping back to ENm010. In all instances the mappable reads
were proportional to the degree of multiplexing, indicating equivalent library
compositionofeach experiment. The heatmapsarescaledasfollows—forFig.1a,
distal (foreskin) FBs: 262–17,467, proximal (lung) FBs: 7–5,846; for Supplemen-
tary Fig. 6, siGFP: 1–100, siHOTTIP: 1–100. Raw data from the 5C experiments
used to generate the binned heat maps in Fig. 1a and Supplementary Fig. 6 can be
found in Supplementary File 1. Raw data are available by request.
portion of HOTTIP as a non protein-coding transcribed region named
ncHOXA13-96 (ref. 12). This region also overlaps expressed sequence tag (EST)
clone AK093987 that was previously observed to be expressed in cancer cell lines
derived from posterior anatomic sites41. 59 and 39 RACE (RLM Race kit, Applied
Biosystems/Ambion) showed full-length HOTTIP RNA to be 3,764 nucleotides,
extending the known transcribed region by more than 1,400 bases. BLAST and
BLAT confirmed that portions of HOTTIP are well conserved in mammals and
even in avians but had no protein coding potential. Full-length HOTTIP RNA
sequence has been deposited at NCBI (accession number GU724873). qRT–PCR
with SYBR Green was conducted as recommended by the manufacturer (Agilent
TTCTTTG (HOTTIP-F) and TGCAGGCTGGAGATCCTACT (HOTTIP-R).
ForSupplementary Fig. 11, endogenous nascent HOTTIP was distinguished from
ectopic HOTTIP expressed from cDNA using primers that spanned intron–exon
Strand-specific RT–PCR. RNA extracted from primary foreskin fibroblasts was
reverse transcribed (SuperScript III, Invitrogen) using combinations of the previ-
ously described HOTTIP-specific primers HOTTIP-F and/or HOTTIP-R as dia-
both HOTTIP-F and HOTTIP-R primers to visually determine strand specificity.
HOTTIP transcript count per cell. The level of HOTTIP transcript per cell was
calculated from the level of HOTTIP in 500,000 cells. Full-length HOTTIP in
pcDNA3.11 was assayed by qPCR using primers HOTTIP-F and HOTTIP-R
at predetermined concentrations in triplicate to generate a linear amplification
PCR value from 500,000 foreskin fibroblasts was determined andplotted, and the
corresponding total molecules of transcript was divided by 500,000 to determine
the approximate number of transcripts per cell.
Single-molecule RNA fluorescence in situ hybridization (RNA-FISH). Single
molecule RNA-FISH was performed as described in ref. 42 with the following
modifications: the amount of hybridization solution per chamber was doubled to
allow for proper coating of the chamber and the amount of glucose-oxidase buffer
was tripled to assist in image acquisition. Images were acquired using an Olympus
RNA interference. Primary foreskin fibroblasts were transfected with siRNAs
targeting HOTTIP and WDR5 using Lipofectamine 2000 (Invitrogen) as per
manufacturer’s instructions. Total RNA was harvested 48–72h later using
TRIzol (Invitrogen) and RNeasy Mini Kits (Qiagen) as previously described34.
For the intronic HOTTIP knockdown experiment in Supplementary Figure 11,
a pool of 10 siRNAs (Supplementary Table 3) targeting intronic regions in
HOTTIP were transfected into foreskin fibroblasts, and RNA isolated as above.
a GFP–chicken HOTTIP fusion transcript was used in a small-scale screen to
identify highly effective shRNA constructs. Eleven shRNAs targeting conserved
regions of chicken HOTTIP were designed and inserted into the pSMP system
(Thermo/Open Biosystems). The reporter construct and shRNA constructs were
cotransfected into Phoenix cells, and HOTTIP transcript levels were analysed via
reducedGFPfluorescenceandbyqRT–PCR. ThreeshRNAsthat wereeffectivein
vitro were then cloned into RCAS vector for studies in chick embryos18.
Chick RNAi. RCAS HOTTIP hairpin and RCAS AP viruses were made by trans-
Fertilized chicken eggs were incubated in a humidified rotating incubator at 37uC
until they reached Hamilton/Hamburger stage 10. Eggs were then windowed to
expose the embryos. After gently removing the vitelline membrane, chicken
embryos were microinjected with RCAS-HOTTIP hairpins and RCAS-AP viruses
and each limb was injected five times. The infected embryos were allowed to
incubate at 37uC and were harvested 2 or 4days after injection to detect viral
infection by immunohistochemistry. Total RNA was extracted from injected fore-
limbs, and RT–PCR analysis was performed 4days after injection. Chicken
embryos were harvested 9days post-injection to carry out whole-mount Alcian
blue staining. A total of 50 animals were injected.
Hairpin sequences for chick HOTTIP were TGCTGTTGACAGTGAGCGAC
CACATCTTCGGGCTGCCTACTGCCTCGGA (2-2-1), TGCTGTTGACAGTG
AGAGAGGAGAGCAGAGCGATGCCTACTGCCTCGGA (3-2-1), and TGCT
HOTTIP overexpression. Full-length HOTTIP and a truncated transcript con-
sisting of exons 1 and 2 (HOTTIPExons 1–2) were cloned into the LZRS vector (gift
of P. Khavari), and then transfected into Phoenix cells (gift of G. Nolan) to
either LZRS-full length HOTTIP (lung), LZRS-truncated HOTTIP (foreskin), or
LZRS-GFP (both lung and foreskin), then passaged over 60days, with periodic
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testing of HOXA and HOTTIP expression by qRT–PCR. These cells were used in Download full-text
the rescue experiments depicted in Supplementary Fig. 11.
2(HOTTIPExons 1–2),andhistoneH2B1mRNAweretranscribed invitrousingT7
polymerase according to manufacturer’s instructions (Promega), denatured, and
refolded in folding buffer (100mM KCl, 10mMMgCl2, Tris pH7.0). GST-tagged
beads (Amersham/GE Healthcare) and blocked with excess yeast total mRNA in
PB100 buffer (20mM HEPES pH7.6, 100mM KCl, 0.05% NP40, 1mM DTT,
in-vitro-transcribed HOTTIP or histone H2B1 mRNA for 45min at room tem-
0.05% NP40, 1mM DTT, 0.5mM PMSF), bound RNAs were extracted and ana-
lysed by qRT–PCR, as previously described.
RNA immunoprecipitation. HeLa-WDR5-Flag cells: 48h after Lipofectamine
2000-mediated transfection of HOTTIP into HeLa WDR5-Flag cells (approxi-
mately 107cells), total protein was extracted as previously described, with modi-
fications44. Briefly, cells were resuspended in Buffer A (10mM HEPES pH7.5,
1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 1.0mM PMSF), lysed in 0.25% NP40,
and fractionated by low speed centrifugation. The nuclear fraction was resus-
pended and lysed in Buffer C (20mM HEPES pH7.5, 10% glycerol, 0.42M KCl,
4mM MgCl2, 0.5mM DTT, 1.0mM PMSF). Combined nuclear and cytoplasmic
(Sigma) or mouse IgG affixed to agarose beads (Sigma) for 3 to 4h at 4uC. Beads
were washed four times with wash buffer (50mM TrisCl pH7.9, 10% glycerol,
using Flag peptide (Sigma), co-immunoprecipitated RNA was extracted and ana-
lysed by qRT–PCR.
incubatedwiththe anti-WDR5(ref. 31)or anti-Sirt6(ab62739,Abcam) antibodies
RNA was extracted and analysed by qRT–PCR.
RNA chromatography. Full-length in-vitro-transcribed HOTTIP RNA was con-
jugated to adipic acid dehydrazide agarose beads as described45. The complexed
and bound proteins visualized by western blotting.
BoxB tethering assay. 293T cells were grown to about 50% confluence in 6-well
encodinga luciferase gene under thecontrol offive tandemGAL4 UAS sites were
co-transfected with plasmids encoding GAL4-WDR5, GAL4-lN (the 22 amino
peptide fused to a C-terminal GFP tag, BoxB (containing five repeats of the lN-
specific 19 nucleotide bindingsite),BoxB fused to full-length LacZ, or BoxB fused
kit (Promega) was used to determinerelative levelsof the luciferase gene product,
following the manufacturer’s protocol.
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