Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.


Author summary
Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including regulation of gene expression. However, the mechanism through which they regulate gene expression is not clearly understood. Here we show that a transcriptional co activator-Psip1 specifically regulates the expression of the lncRNA Hottip. Moreover, using multiple approaches, including lncRNA depletion, genetic manipulation of Hottip locus, transcriptional activation and premature termination of lncRNA transcript along with RNA localization, we demonstrate that Hottip lncRNA regulates expression of neighbouring Hoxa genes.

Introduction
The mammalian genome encodes~10,000 long noncoding RNAs (lncRNAs) [1]. Although very few of these molecules have been functionally characterised, a small number have been shown to function by binding to various protein complexes to regulate gene expression [2][3][4][5][6]. Some lncRNAs have been reported to affect gene expression in trans [7,8], whereas others, such as Kcnq1ot1, Xact, Xist and Tsix, function in cis (reviewed in [9]). Other lncRNAs likely function in the cytoplasm through binding to other regulatory RNAs, e.g. miRNAs [10].
It has also been difficult to distinguish whether lncRNA function is conferred by the process of transcription or by the RNA molecule itself. Concerns have been raised with respect to limitations and discrepancies in various methodologies used to study lncRNA function [11][12][13]. Contrasting conclusions have often been reached when comparing knockdown and knockout studies of lncRNA loci-e.g. HOTAIR, MALAT1 and Halr [14][15][16][17].
With the exception of relatively well characterized lncRNAs like Xist [18], H19 [19,20] and Kcnq1ot1 [21,22], many recently described lncRNAs lack genetic evidence to support their function in vivo. Indeed, recent efforts to phenotype mouse knockouts for 18 lncRNA genes identified only 5 with strong phenotypes [23]. With the list of lncRNA loci with unknown function increasing, there is a pressing need to rigorously dissect the functional mechanisms of individual lncRNA loci. Additionally, most research has focused on the downstream functions of lncRNAs and, with the exception of lncRNAs involved in imprinting and dosage compensation, little is known about the transcriptional regulation of lncRNAs themselves. Compared to lncRNA sequences, the promoters of lncRNA genes are conserved, and are enriched for homeobox domain containing transcription factor binding sites [24], which suggests lncRNA expression is a regulated process.
Mammalian Hox loci are important model systems for the investigation of lncRNA functions. Expression of many noncoding RNAs within Hox clusters is tissue specific [25][26][27][28][29], and have been linked to the regulation of Hox mRNA genes [7,14,30,31]. At the Hoxa cluster, the lncHoxa1/Halr-also known as Haunt is located~50 kb away from 3' end of HOXA ( Fig 1A) and has been shown to repress HOXA1 expression in cis [32]. Importantly, a recent study demonstrated that Haunt lncRNA plays a distinct role as a repressor while its DNA sequence functions as an enhancer for HOXA genes [15]. HOTAIRM, located between HOXA1 and HOXA2, is expressed antisense to coding HOXA genes, and is implicated in retinoic acid induced activation of HOXA1 and HOXA4 during myeloid differentiation [33]. HOTTIP lncRNA is transcribed in an antisense direction from the 5' end of HOXA13 (Fig 1A), and is reported to be important for targeting MLL through interaction with WDR5 to maintain posterior (5') HOXA expression in distal tissues [3].
PC4 and SF2 interacting protein (Psip1), also known as LEDGF, has been suggested to play an important role in regulation of Hox genes [34]. We have recently demonstrated the role of the p75 isoform of Psip1 (Psip1/p75) in recruiting an Mll complex to expressed Hox genes [35]. The alternatively spliced short isoform of Psip1 (Psip1/p52) lacks the C-terminal Mll or integrase binding domain (IBD), but shares the chromatin binding PWWP and AT hook like domains at the N-terminus. Psip1/p52 binds to H3K36 trimethylated (H3K36me3) nucleosomes via the PWWP domain and can modulate alternative splicing by recruiting splicing factors to H3K36me3 [36].
Here, we show that Psip1/p52, but not Psip1/p75 regulates the expression of the lncRNA Hottip, which is located at the 5' end of the Hoxa locus and transcribed in an antisense direction away from Hoxa13. We show that the Hottip RNA binds to, and is required for, activation of genes at the 5' end of Hoxa establishing a firm role for a lncRNA molecule in the regulation of gene expression in cis. This also adds a new role for Psip1/p52 in RNA-based processes.  [35]. Annotated noncoding transcripts (grey, top) and Hox gene transcripts (black) are shown below. (n = 2 biological replicates). Genome co-ordinates are from the mm9 assembly of the mouse genome. Direction of transcription for Hoxa13 and Hottip genes are indicated with arrow below. (B) Mean (± s.e.m) expression, assayed by RT-qPCR and normalized to Gapdh, of

Psip1 is required for expression of lncRNA Hottip
In mammals, the active state of Hox genes is maintained by Compass-like complexes containing the MLL (Mix lineage leukemia) histone H3K4 methyltransferases. Hox repression is maintained by Polycomb (PcG) complexes [37]. We recently demonstrated that the transcriptional co-activator Psip1/p75 and Mll co-occupy expressed Hox genes, and that loss of Psip1 leads to reduced Mll1 (and Mll2) occupancy at active Hox genes [35]. Most strikingly, at the extreme 5' end of Hoxa, where the Hottip lncRNA is located [3], Mll binding is completely lost in Psip -/-MEFs compared to wild type (WT) (Fig 1A). Reduced Mll1 is accompanied by concurrent loss of H3K4me3 and Menin-a common component of Mll1 and Mll2 Compass-like complexes [38]. We noted that absence of Psip1 results in complete loss of expression of the lncRNA Hottip and reduced expression of Hoxa13, which is located adjacent to Hottip at the 5' end of Hoxa and which has previously been described as one of the target genes of Hottip ( Fig  1B) [3]. In contrast, other Hottip target genes-Hoxa9, a10, and a11 [3] are up-regulated in Psip1 -/-MEFs despite the loss of Hottip expression ( Fig 1C) [35]. Nascent run-on transcription analysis shows that these effects occur at the level of transcription ( Fig 1C). Together with the binding of Psip1 to the expressed 5' part of Hoxa (Fig 1A), these results suggest that Psip1 might function as a transcriptional coactivator to regulate expression of the Hottip lncRNA.
Depletion of Psip1/p52 and Hottip leads to reduced expression of 5' Hoxa genes Stable rescue of Psip -/-MEFs with the p52 isoform of Psip1 led to an increase in expression of posterior Hoxa genes (Fig 2A) suggesting a role for the short Psip1 isoform in this regulation. To confirm this finding in a different cell type we analysed Psip1-mediated Hoxa expression in a limb bud mesenchymal cell line (14fp) which retains the distal limb-specific expression pattern of posterior or 5' Hox genes [39]. Psip1 is expressed at high levels in the distal limb buds of mouse embryos, where Hottip and 5' Hoxa genes are also highly expressed (S1A Fig) [3]. Moreover, Hoxa13 expression is required for patterning of the distal limb [40].
To identify which isoform of Psip1 regulates Hottip in the limb bud cell line we knocked down Hottip and also the two separate isoforms of Psip1 using two independent sets of lentiviral shRNAs each specifically targeting the 3' UTR of Psip1/p52, the C-terminus of Psip1/p75 and Hottip RNA. Knockdown efficiency was confirmed by RT-qPCR analysis (Fig 2B and S1B  Fig) and by immunoblotting for Psip1 isoforms (Fig 2C). Knockdown of Psip1/p75 had no significant affect on Hottip or Hoxa genes in these cells. However, specific knockdown of Psip1/ p52 led to down-regulation of 5' Hoxa genes-Hoxa13, a11 and a10, with Hoxa13 expression being the most strongly abrogated (Fig 2B). Knockdown of p52 also strongly downregulated Hottip expression (Fig 2B and 2D) and this was rescued by expression of a shRNA-resistant p52 cDNA (Fig 2D). Knockdown of Hottip had an almost identical affect on 5' Hoxa expression as p52 knockdown ( Fig 2B) and is consistent with the reported effects of HOTTIP knockdown in human foreskin fibroblasts [3]. These data suggest that it is the p52 isoform of Psip1, not p75, that specifically activates Hottip lncRNA transcription. Moreover, these data support an earlier report that the Hottip lncRNA is involved in maintaining the active chromatin domain at 5' Hoxa genes [3]. Log2 mean (± s.e.m) relative expression, assayed by RT-qPCR and normalized to Gapdh, of Hoxa genes, along with Psip1/p52, Psip1/p75, and total Hottip (exon 2) transcript, in limb cells transduced with shRNAs targeting p52 (red bars, p52 sh1) p75 (green bars, p75 sh1) and Hottip (black bars, Hottip sh1) relative to cells transduced with a mammalian non-targeting sh RNA (control) (n = 3 biological replicates). * p < 0.05, ** p <0.01. (C) Immunoblotting of limb cells after shRNA knockdown of p52 and p75 Psip1 isoforms with Psip1 antibody (A300-847a) which recognizes both p52 and p75 [36]. β-actin served as loading control. Two different sets of shRNAs (sh1 in (a) and sh2 in S1 Fig) were used for knockdown along with a mammalian non-targeting shRNA as control (control_sh). Knockdown of p52, p75 and Hottip using independent lentiviral shRNAs (sh2) confirms that mis-regulation of Hox genes is not due to off-target effect of shRNAs (S1B Fig). (D) Mean log2 expression of Hottip, in limb cells transduced with sh RNAs targeting p52 (red bars, p52 sh1) and those cells rescued transiently with shRNA resistant p52 cDNA (green bars, p52 sh1 p52res). Fold change in expression was normalized to Gapdh, relative to mammalian non-We found a significant reduction in total Hottip RNA levels in the p52 knockdown cells (Fig 2B), which shows that reduced Hottip RNA levels are not simply due to the known effect of Psip1/p52 on RNA splicing [36].

Mll1 occupancy over Hoxa cluster is altered upon p52 & Hottip knockdown
It has been suggested that Hottip has a role in maintaining an MLL complex through interaction with the WDR5 component [3]. Consistent with this, ChIP showed that Mll1 occupancy was significantly reduced across posterior Hoxa genes in limb bud cells upon knockdown of p52 or Hottip compared to control knockdown (Fig 2E), Intriguingly, whilst Mll1 was completely lost from Hoxa13 upon depletion of p52 and Hottip (Fig 2E), it was gained at 3' Hoxa genes (Hoxa1 -a6), concomitant with the increase in expression of these 3' Hoxa genes upon p52 or Hottip depletion ( Fig 2B). This redistribution of Mll is consistent with the redistribution of H3K4me3 and Menin across Hoxa and Hottip loci in Psip1 -/-MEFs (Fig 1A), although the causal mechanism is not known.

Deletion of Hottip leads to reduced expression of posterior Hoxa genes
Most lncRNA depletion studies are done by si/sh RNA mediated knockdown, but the conclusions reached have often been different from those after genetic deletion of the loci encoding the lncRNAs [14][15][16]. We therefore used two pairs of guide RNAs with Cas9 nickase (Cas9n) to delete the gene body of Hottip (HottipΔ) in limb mesenchymal cells, leaving the Hottip promoter intact (Fig 3A). qRT-PCR of Hoxa genes showed a significant reduction in expression of Hoxa13, a11 and a10 in homozygous HottipΔ cells (Fig 3B). Consistent with Psip1 and Hottip knock down studies (Fig 2B), expression of 3' Hoxa genes, such Hoxa2, a6 and a7 increased in HottipΔ compared to WT cells. It is possible that effects on 3' Hoxa genes are due to crossregulation of Hox genes by Hox transcription factors [41].
Hottip RNA is localized at posterior Hoxa genes To find the direct genomic targets of Hottip lncRNA in limb cells, we performed chromatin isolation by RNA purification (ChIRP) [42] using 11 biotinylated antisense oligo pools covering the entire length of Hottip. qRT-PCR analysis of ChIRPed RNA showed specific enrichment for Hottip RNA (Fig 3C). qPCR analysis of Hottip ChIRPed DNA showed specific enrichment of Hottip RNA over the promoters of Hoxa13, and a11 in WT cells. Analysis in HottipΔ cells confirmed the specificity of the Hottip ChIRP (Fig 3D). Hottip RNA was undetectable across more 3' Hoxa genes (a9, a7, a1), demonstrating that misregulation of 3' Hoxa genes in the absence of Hottip (Figs 2B and 3B) is a secondary event, which does not involve direct binding of Hottip.

Induction of Hottip lncRNA is sufficient to activate posterior Hoxa genes
It is possible that reduced expression of posterior Hox genes in HottipΔ cells is due to loss of cis -regulatory elements located within the deleted region, rather than loss of the Hottip RNA per se. Hottip is known to function at the site of its synthesis (in cis) and it fails to activate target genes when expressed ectopically from a retroviral construct [3]. Therefore, we targeting shRNA (control) (n = 3 biological replicates). (E) Mean Log2 ChIP/input across Hoxa cluster for Mll1 from limb cells transduced with control shRNA (Control_Sh), shRNA targeting p52 (p52 sh1) and Hottip (Hottip sh1) as described in (B) & (C). Genome co-ordinates are from the mm9 assembly of the mouse genome.
We have previously shown that targeted recruitment of an ectopic activator (Vp16) to silent loci in murine ES cells (mESCs) can overcome this repression [43]. Unlike human HOTTIP which is transcribed bi-directionally from the HOXA13 CpG island promoter (Fig 4A) [3], the mouse Hottip promoter is~2 kb away from the TSS of Hoxa13 which allowed us to recruit  Psip1/p52 regulates Hox genes through lncRNA Hottip dcas9-Vp160 (Vp16 x10) [44][45][46][47] specifically to the promoters of either Hottip or Hoxa13 ( Fig  4A). CRISPR dCas9 mediated transcriptional activation has been shown to be ineffective when guides are targeted >1kb from TSS [44] suggesting that we should be able to direct activation specifically to Hottip or Hoxa13 using this approach.
Agilent expression microarray and RT-qPCR analysis showed specific up-regulation of posterior (a13, a11 and a10), but not anterior Hoxa and Hoxd genes upon dcas9-VP160 mediated Hottip activation in mESCs, relative to transfection with dCas9 recruitment alone (no VP160) (Fig 4B and 4C). In contrast, specific recruitment of dCas9-VP160 to the Hoxa13 promoter led to up regulation of only Hoxa13, while expression of other Hoxa genes was unaltered (Fig 4B). Furthermore, recruitment of dCas9-VP160 to either Hox13 or Hottip did not perturb the pluripotency network (Fig 4B) suggesting that the undifferentiated phenotype of the mESCs was not disrupted. Finally, recruitment of dCas9-VP160 to the Hottip promoter in HottipΔ 14fp cells led to only a modest upregulation of Hoxa13 compared to WT cells (Fig 4D), pointing to the importance of full length Hottip RNA transcription in the regulation of Hoxa genes.

Hottip RNA is indispensable for 5' Hoxa expression
To distinguish the requirement for the Hottip lncRNA molecule from the act of lncRNA transcription at the Hottip locus, for up-regulation of 5' Hoxa genes, we used CRISPR-Cas9-mediated homologous recombination to insert a 49 bp synthetic polyadenylation (polyA) cassette [48] 47 bp downstream of the Hottip transcription start site (TSS) in 14fp cells (Fig 5A and  5B). Insertion of this polyA cassette should cause early cleavage of the nascent lncRNA transcript while preserving the promoter, and cis elements within the Hottip genomic locus. RT-qPCR analysis in three independent knockin lines and two wild-type (WT) clones demonstrated that spliced Hottip RNA was strongly reduced in all three polyA knockin lines (pA1, pA2 and pA3) compared to WT (Fig 5C). Importantly Hoxa13 and a11 mRNA levels were significantly reduced in all three pA lines compared to WT.
To verify this effect in vivo, we also injected Cas9 and guideRNAs into single cell zygotes to generate mouse embryos with a premature polyA signal inserted at Hottip (S1C Fig). Consistent with the results in the 14fp cell line, RT-qPCR analysis of polyA knockin 12.5 dpc embryo showed reduced expression of Hottip, Hoxa13 and Hoxa11 but not Hoxc13 -a posterior Hox gene from different chromosome (S1C Fig). This suggests that it is the full length Hottip RNA itself that is involved in Hoxa13/a11 regulation.

Discussion
Our findings are compatible with a model in which Hottip lncRNA regulates posterior Hoxa gene transcription in cis (Fig 5D)-likely through an Mll complex. We have previously shown that the longer p75 isoform of Psip1 binds directly to Mll through its MLL or integrase binding domain (IBD) and recruits Mll to active Hox clusters [35]. Here we have demonstrated that the shorter isoform Psip1/p52 -which lacks the C-terminal Mll1 binding domain of p75-controls posterior Hoxa genes by activating the expression of Hottip lncRNA, a mechanism quite distinct from p75. We show that the Hottip lncRNA itself is required to maintain active expression of 5' Hoxa genes, possibly by maintaining a stable Mll1 complex at the 5' end of Hoxa gene cluster. The mechanism through which Hottip RNA specifically localizes to 5' Hoxa genes in cis is not clear and needs further investigation.
By inserting a polyadenylation cassette at the 5' end of Hottip, we show the importance of Hottip RNA for Hoxa13 expression in both cell lines and in vivo in mouse embryogenesis. Similarly, Hottip is upregulated in several cancers where its expression also correlates with increased Hoxa13 [49][50][51]. Recently, two micro RNAs miR-192, miR-204 have been demonstrated to Noncoding transcription at enhancer elements has been associated with enhancer activity [53,54]. However, most enhancer RNAs (eRNAs) are degraded by exosomes, suggesting that at distal regulatory elements the act of transcription itself could be sufficient for the enhancer activity [55][56][57]. An enhancer -like function of lncRNAs has been demonstrated in some cases including HOTTIP [3,58]. However, an increasing body of evidence suggests that the function of many lncRNA genes in regulating genes in cis does not require the lncRNA molecule itself. Instead their effect is mediated by enhancer-like activity of underlying DNA elements in the lncRNA locus, the act of transcription and/or splicing of lncRNAs [59][60][61][62].
The recent controversies in the lncRNA field demand thorough investigations to distinguish the role of lncRNA molecules from enhancer-like function of the DNA elements which encode them, and from the process of transcription and splicing of these loci. Our studies presented here show how these facets of lncRNA regulation and function can be dissected at one well-studied lncRNA locus. With the ever increasing number of lncRNAs annotated in genomes using high-throughput sequencing technologies, the list of these transcripts with unknown mechanisms of upstream transcriptional regulation and downstream functional mechanism is growing and there will be the need to develop more high-throughput methods for the rigorous testing of lncRNA function and mechanism of action.

Ethics statement
Cervical dislocation was used as a euthanasia method and all mouse experiments were performed under the Animals (Scientific Procedures) Act 1986' and were approved by the University of Edinburgh ethical committee (TR-38-16) and performed under UK Home Office license number PPL 60/4418.

Cell lines
Psip1 -/and its corresponding WT MEFs [35,63] were a kind gift from Prof. Alan Engelman (Dana-Farber Cancer Institute, USA). Limb mesenchymal cells (14fp) isolated from the posterior mesenchyme of E11.5 mouse embryos from an Immortomouse (H-2kb-tsA58) × CD1 cross, are as previously described[39] and were a gift from Robert Hill (MRC Human Genetics Unit, University of Edinburgh). mES cells (E14) were cultured as previously described [64]. Psip1/p52 rescue experiment in Psip -/-MEFs is previously described [35]. are shown as RT-Fp and RT-Rp. (B) Genotyping PCR from the DNA isolated from the wild type (WT1, WT2) and polyA knock-in (pA1, pA2, and pA3) 14fp lines. (C) RT-qPCR data showing mean (± s.e.m of three technical replicates) fold change in expression of Hottip, Hoxa13, a11, a10, a9 and a7 in wild type (WT) and three polyA knock-in 14fp lines. * p < 0.05, ** p < 0.01. (D) Working model summarising the results from various experiments. Wild type cells with normal level of posterior Hoxa expression and occupancy of Hottip RNA at Hox genes (i). In Psip1 -/-MEFs the expression levels of Hottip, posterior Hoxa genes and bound Mll levels are reduced (ii). Knockdown of Psip1/p52 or Hottip reduced expression of posterior Hoxa genes with corresponding reduction of Mll levels at these sites (iii). Deletion of Hottip leads to similar effect as depletion of Hottip by shRNAs (iV). Artificial activation of Hottip in mESCs leads to increased expression of target Hoxa genes (V). Premature termination of Hottip transcript leads to reduced expression of target Hoxa genes (Vi). The effects of each approach in changing the DNA element, lncRNA and the process of transcription are indicated (right).

RT-qPCR
Reverse transcription followed by quantitative PCR (RT-qPCR) was performed as described previously [35]. Briefly, RNA was treated with Turbo DNA Free kit (ThermoFisher Scientific) and cDNA was prepared using superscript II reverse transcriptase (ThermoFisher Scientific) using random primers. All qPCRs were performed with three biological or technical replicates in a LightCyler 480 (LC480, Roche) or CFX96 (Biorad), and the data was normalized to Gapdh. Details of the oligos are given in the S2 Table. Whole mount RNA in situ on mouse embryos RNA in situ hybridization for mHottip in 10.5 dpc mouse embryos were performed as previously described [65]. Details of oligos used to PCR amplify the Hottip cDNA including T7 (sense) and T3 (Antisense) promoter sequences are given in S3 Table. ChIP, antibodies and data analysis ChIP was performed as described previously [35], using antibodies for Mll1 (Active Motif 61295, 61296), ChIP DNA was hybridized to a custom Hox array and data was normalized as described previously [35].

Chromatin Isolation by RNA Purification (ChIRP)
Anti-sense oligo probes tiling the mouse Hottip RNA were designed using the web tool from Stellaris FISH Probe Designer (https://www.biosearchtech.com/support/education/stellarisrna-fish) Biosearch Technologies, CA, USA). Eleven biotinylated oligos were synthesized by Sigma-Aldrich (S6 Table). ChIRP was performed in limb mesenchymal cells as described previously [42]. RNA was isolated from 20% of the ChIRPed beads and used for RT-qPCR for Hottip, 7sk and Gapdh specific primers and rest of the sample was used to purify DNA and perform qPCR for Hoxa genes. Primer details are given in S2 Table. CRISPR mediated deletion of Hottip Guide RNAs were designed using the Zhang laboratory web tool (http://crispr.mit.edu). Paired guide RNAs (gRNAs) (S4 Table) were designed to target the murine Hottip genomic locus 50bp beyond the TSS and before the transcription end site (Fig 3A). gRNAs were cloned into the D10A nickase mutant version of cas9 (cas9n) containing pSpCas9n(BB)-2A-GFP (PX461) [66]. A pool of four gRNA containing plasmids were transfected into mouse limb-bud mesenchymal cells (14fp) using FuGENE HD transfection reagent and FACS sorted 48 hours after transfection for GFP + cells. Homozygous deletion of Hottip was confirmed by PCR and Sanger sequencing, primers used are given in S2 Table. dCas9-mediated activation of Hottip and Hoxa13 in ES cells Five guide RNA plasmid pools targeting the promoters of Hottip and Hoxa13 (S5 Table) were designed as above and cloned into pSLQ1371 [56,67]. These gRNA plasmids encoding mCherry and puromycin resistance were co-transfected with a plasmid encoding dCas9-VP160 (pAC94-pmax-dCas9VP160-2A-puro, Addgene plasmid number 48226) [47]. 24hrs after transfection, transfected mES cells were selected by addition of 2μg/ml puromycin for another 24 hrs. RNA was extracted 48hrs after transfection, RT-qPCR was performed as described above. Microarray gene expression analysis performed according to the manufacturer's protocol (Agilent Technologies). Plasmids containing non-targeting guide RNAs and dCas9 alone (dcas9Δ) served as controls.

dCas9-mediated activation of Hottip in 14fp cells
Wild type and HottipΔ 14fp cells were transfected with Hottip gRNA plasmid pools similar to ES cells and FACS sorted for mCherry positive cells 24 hrs after transfection. Transfected mCherry positive cells were seeded to cell culture flasks to recover for another 24 hrs, 48 hrs after transfection cells were harvested and RNA was isolated using Trizol and RT-qPCR was performed.  Table) were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (a gift from Feng Zhang (Addgene plasmid 62988)), which is designed to insert a synthetic polyA signal sequence into the Hottip genomic locus 47 bp after the TSS (Fig 5A). gRNA containing plasmids were co-transfected along, with a repair template (S7 Table) synthesized as a 199bp single-stranded Ultramer oligo (IDT) bearing the desired sequence change, into limb cells using lipofectamine 2000 transfection reagent. 24 hours after transfection puromycin resistant cells were selected for another 48 hours and plated at 2500 cells/100mm plates. On day 10 colonies were picked and plated in duplicate into 96 well plates. Genomic PCR ( Fig  5B) and Sanger sequencing confirmed three polyA knockin (pA) clonal lines with homozygous insertions of the polyA cassette into exon 1 of Hottip. Primers used for genotyping PCR sequencing and RT PCR are given in S2 Table. CRISPR mediated insertion of polyA sites into Hottip in mouse embryo