A conserved <i>HOTAIRM1-HOXA1</i> regulatory axis contributes early to neuronal differentiation

HOTAIRM1 is unlike most long non-coding RNAs in that its sequence is highly conserved across mammals. Such evolutionary conservation points to it having a role in key cellular processes. We previously reported that HOTAIRM1 is required to curb premature activation of downstream HOXA genes in a cell model recapitulating their sequential induction during development. We found that it regulates 3’ HOXA gene expression by a mechanism involving epigenetic and three-dimensional chromatin changes. Here we show that HOTAIRM1 participates in proper progression through the early stages of neuronal differentiation. We found that it can associate with the HOXA1 transcription factor and contributes to its downstream transcriptional program. Particularly, HOTAIRM1 affects the NANOG/POU5F1/SOX2 core pluripotency network maintaining an undifferentiated cell state. HOXA1 depletion similarly perturbed expression of these pluripotent factors, suggesting that HOTAIRM1 is a modulator of this transcription factor pathway. Also, given that binding of HOTAIRM1 to HOXA1 was observed in different cell types and species, our results point to this ribonucleoprotein complex as an integral part of a conserved HOTAIRM1-HOXA1 regulatory axis modulating the transition from a pluripotent to a differentiated neuronal state.</p

Additional file 1: Figure S1. of Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

Toxicity of NU7441, KU-0060648 and Scr7 on the cell lines used in this study. A The 293/TLR line was exposed to increasing concentrations of NU7441, KU-0060648, and Scr7 for 5 days at which point the viability of the cells was determined. Viability is plotted relative to vehicle controls. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *P ≤0.05; **P ≤0.01; ns, not significant. B Viability of Arf −/− MEFs exposed to NU7441 or KU-0060648. Experiments were performed as for 293/TLR cells in (A). (PDF 371 kb

Regulatory <i>HoxA</i> contacts are independent of enhancer activity.

<p><b>A.</b> Analysis of e1 and e5 activity in <i>Shh−/−</i> limbs. Upper panel: Scheme representing loci bound by Gli3R (blue bars) along the <i>HoxA</i> regulatory region. The IR50 transgene used as control contains the 50 kb intergenic region to <i>Hoxa13</i> and <i>Evx1</i>, which includes e1. Active genes are shown in red, and arrows indicate the position of promoters and transcription direction. Lower panel: LacZ staining showing e5 (a–d) and e1 (e–h) transcriptional activity, in wt (a; e) and <i>Shh−/−</i> embryos (b–d; f–h). <b>B.</b> Long-range e5 interaction with <i>Hoxa13</i> is independent of its activity. The physical proximity between the <i>Hoxa13</i> gene and e5 was measured by 3C. The position of e5 is highlighted in green. Interaction frequency were measured compared to a BAC 3C library as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#s4" target="_blank">Materials and Methods</a>. Error bars correspond to the standard error of the mean. <b>C.</b> Physical contacts between the <i>HoxA</i> cluster and the upstream genomic region measured by 5C-seq in wild-type distal limb (<i>top</i>), <i>Shh−/−</i> mutant distal limb (<i>middle</i>), and the head (<i>bottom</i>) of mouse embryos. 5C data is presented in the form of heatmaps according to color scales as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen-1004018-g002" target="_blank">Figure 2</a>. The limb-specific interaction pattern between enhancers and the 5′ <i>HoxA</i> genes are similar in <i>Shh−/−</i> (<i>middle</i> panel) and wt distal limb buds (<i>top</i> panel) albeit with some interaction frequencies slightly reduced. Dotted lines delineate the regions containing the enhancers bound by Gli3R (e3, e5 and e16). Gli3R sites are represented with blue bars. Brackets on the left hand side of each heatmap show the area containing <i>Hoxa9</i>, <i>a10</i>, <i>a11</i>, and <i>a13</i>. Green arrows indicate the chromatin fragments containing the <i>Hibadh</i> and <i>Jazf1</i> promoters (p). Restriction fragments corresponding to enhancer e6–8, 12, and 19 are not shown in the heatmaps as they fell into regions that were not amenable to 5C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#s4" target="_blank">Materials and Methods</a>).</p

Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of <i>HoxA</i> Genes in Developing Limbs

<div><p><i>HoxA</i> genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of <i>bona fide</i> transcriptional enhancers controlling <i>HoxA</i> genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with <i>HoxA</i> genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of <i>HoxA</i> and <i>HoxD</i> regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.</p></div

Extensive clustering of genes and enhancers highlights a complex regulation network in distal limbs.

<p><b>A,B.</b> 5C interaction matrix of the <i>HoxA</i> cluster and its upstream regulatory region in distal limb (<b>A</b>) and head (<b>B</b>). The 5C data was generated by 5C-seq using tissues from E12.5 embryos, and is presented in the form of heatmaps according to color scales as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen-1004018-g002" target="_blank">Figure 2</a>. Heatmaps above the linear diagram of the genomic region show interaction frequencies for each restriction fragment, irrespective of their size. Heatmaps at the bottom show the mean interaction frequencies per 20 kb DNA fragment and were obtained from binning and smoothing of the 5C raw data. Expressed genes within the region are colored in red. The yellow and green shading links the genomic position of <i>HoxA</i> and <i>Evx1</i> genes, and the enhancer clusters to the corresponding areas in heatmaps. Black arrows point to interactions between the gene sub-TADs and enhancer sub-TADs. White lines delineate the TAD and sub-TADs therein. Dashed white lines are drawn to highlight the sub-TAD interactions. <b>C.</b> Topological organization of the <i>HoxA</i> cluster and <i>Evx1</i>. Genes are organized in three sub-TADs in the limb (<i>top</i>). Interaction enrichment in head tissues compared to the limb (<i>bottom</i>) shows significant increase in interaction between the gene sub-TADs in the head. Smoothing was performed based on distance (8 kb) and heatmap intensities represent the mean of interaction frequency for each 8 kb window. <b>D.</b> Extensive limb-enriched interactions between distal <i>HoxA</i> enhancers suggest that a physical network regulates 5′ <i>HoxA</i> genes in the limb. The interaction matrix of the region containing enhancer e10 to e18 is shown in the form of a heatmap. Limb-enriched contacts are shown in red according to the color scale as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen-1004018-g002" target="_blank">Figure 2</a>.</p

Model illustrating how genome topology underlies the tissue-specific regulation of <i>HoxA</i> genes.

<p>The <i>HoxA</i> cluster is partitioned between two TADs (light blue), physically segregating <i>3′HoxA</i> from 5′<i>HoxA</i> genes in a mostly cell-type independent manner. In contrast, the sub-TAD interaction pattern is drastically different in the limb (<b>A</b>) compared to the head (<b>B</b>). Limb enhancer sub-TADs (dark blue) interact with each other and with gene-sub-TADs in distal limb but not head tissue. Enhancer and gene interactions occur between sub-TADs from the same TAD (5′<i>HoxA</i> containing TAD) but not with 3′<i>HoxA</i> genes that are located in the adjacent TAD. The limb-specific sub-TAD interactions create a platform architecture controlling <i>HoxA</i> expression by the remote distal limb enhancers upon enhancer activation by transcription factors. The schemes of the chromatin conformation were designed assuming cellular homogeneity within each tissue.</p

Candidate limb enhancers reside on the telomeric side of the <i>HoxA</i> cluster.

<p><b>A, B.</b> Distal limb enhancer activity lies upstream of the <i>HoxA</i> cluster and does not require sequences within it. Expression of the Neomycin and Hygromycin reporter genes flanking the cluster were analyzed by whole mount <i>in situ</i> hybridization on E11.5 embryos. In embryos where the <i>HoxA</i> cluster is intact (<b>A</b>), expression of the upstream Hygromycin reporter was detected in the distal part of the limb while downstream neomycin transcripts were not. TKNeo: minimal thymidine kinase promoter upstream of Neomycin reporter gene. PGKHygro: minimal phosphoglycerate kinase-1 promoter upstream of Hygromycin reporter gene. Arrow above the <i>HoxA</i> cluster diagram shows transcription direction. <b>B.</b> Neomycin expression after deletion of the cluster and PGKHygro by recombination of <i>loxP</i> sites flanking the reporter genes shows that sequences within the cluster are not required for distal limb enhancer activity. <b>C.</b> Distal limb cells analyzed in this study express 5′ <i>HoxA</i> genes (<i>Hoxa9</i> to <i>a13</i>). <i>HoxA</i> gene expression in developing limbs is illustrated on the <i>left</i>. The dotted line indicates the area micro-dissected to collect distal limb cells for analysis. Stylopod: upper arm, zeugopod: lower arm, mesopod: wrist, autopod: hand. <b>D.</b> The position of candidate enhancer sequences was identified by ChIP-seq. Proteins known as being enriched at active enhancers (RNAP2, Med12, p300) and the H3K27Ac histone mark was examined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#s4" target="_blank">Materials and Methods</a>. The y-axis corresponds to “reads per million” except for the p300 data where the number of sequence reads is shown. Colored rectangles below each track indicate the position of significant peaks. The position of candidate enhancers (e1 to e19) is highlighted in green below the genomic region characterized, where transcriptionally active genes are in red and arrows indicate transcription direction. Sequence conservation in the chicken is shown on the bottom.</p

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

<div><p>The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the <i>in vivo</i> target specificity of Cas9 is poorly understood and most studies rely on <i>in silico</i> predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the <i>Trp53</i> locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to ^5′NGG^3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. <i>In vitro</i> analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.</p></div

Number and Percentage of Total, Aligned, Duplicate, and Processed Reads Obtained from Chromatin Immunoprecipitates or Whole Cell Extracts (WCE) of Arf^−/− MEFs infected with pQdmCiG, pQdmCiG/sgp53-1, or pQdmCiG/sgp53-3.

<p>*After mapping and removal of duplicates.</p><p>Number and Percentage of Total, Aligned, Duplicate, and Processed Reads Obtained from Chromatin Immunoprecipitates or Whole Cell Extracts (WCE) of Arf^−/− MEFs infected with pQdmCiG, pQdmCiG/sgp53-1, or pQdmCiG/sgp53-3.</p