Chemical shift assignments of mouse HOXD13 DNA binding domain bound to duplex DNA

The homeobox gene (Hoxd13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of proteins that control embryonic morphogenesis. We report NMR chemical shift assignments of mouse Hoxd13 DNA binding domain bound to an 11-residue DNA duplex (BMRB No. 25133)

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules

<i>LncRNA-HIT</i> Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes

<div><p>Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), <i>LNCRNA-HIT</i> in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, <i>LncRNA-HIT</i> was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of <i>LncRNA-HIT</i>-p100/CBP complexes by ChIRP-seq revealed <i>LncRNA-HIT</i> associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by <i>LncRNA-HIT-</i>p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in <i>LncRNA-HIT</i> or p100 transcripts revealed a significant decrease in expression of many of the <i>LncRNA-HIT</i>-associated loci. <i>LncRNA-HIT</i> siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the <i>LncRNA-HIT</i> siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that <i>LncRNA-HIT</i> is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using <i>LncRNA-HIT</i> and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.</p></div

Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.

<p>Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.</p

<i>LncRNA-HIT</i> recruits a complex of p100 and CBP in the limb.

<p><b>(A-D)</b><i>Snd1</i> which encodes p100 is expressed in many of the same regions as <i>LncRNA-HIT</i> including the limbs at E11-13.5, spinal cord, and genital tubercle. FL = forelimb, HL = hindlimb, MX = maxillary component of the first branchial arch, SC = spinal cord, P = pinnae. <b>(E)</b> Western blot analysis using antibodies specific for CBP and p100 confirm the presence of both proteins in cell lysates from E 11.5 limb buds. <b>(F)</b> Streptavidin-mediated precipitation of biotinylated <i>LncRNA-HIT</i> and <i>U1</i> RNA transcripts reveals co-precipitation of CBP and p100 from limb mesenchyme, confirming recruitment of both proteins by the lncRNA in the limb mesenchyme. <b>(G)</b> RIP analysis of p100 and CBP from the limb mesenchyme reveals co-precipitation of endogenous <i>LncRNA-HIT</i> transcript, confirming that CBP, p100, and <i>LncRNA-HIT</i> are present in a complex in the limb bud mesenchyme. Values represent the mean fold enrichment of <i>LncRNA-HIT</i> after precipitation with antibodies specific for CBP, p100 from three independent assays. Control precipitations were performed in parallel using murine IgG. Bars represent the standard deviation of the mean from the three independent assays.</p

Expression analysis of the lncRNA <i>LncRNA-HIT</i>.

<p><b>(A)</b><i>In situ</i> hybridization using an antisense <i>LncRNA-HIT</i> riboprobe detects the transcript as early as E 10.5 in the distal limb which expands throughout the limb bud at E 11.5 <b>(B)</b>. <b>(C)</b> <i>In situ</i> hybridization using a sense orientation <i>LncRNA-HIT</i> riboprobe detects no <i>LncRNA-HIT</i> transcripts, confirming the unidirectional transcription of <i>LncRNA-HIT</i> in the same orientation as the 5’ HoxA genes in the developing limb. <b>(D and E)</b> Analysis of <i>Hoxa13</i> expression shows a high level of overlap with <i>LncRNA-HIT</i> in the distal limb. <b>(F-H)</b> Analysis of <i>Hoxa9-Hoxa11</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i> in the limb bud. <b>(I-K)</b> Analysis of <i>Hoxd11-Hoxd13</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i>. <b>(L)</b> <i>LncRNA-HIT</i> is also expressed in the developing genital tubercle, gut epithelium, urogenital sinus, spinal cord, and vertebral bodies at E 13.5. <b>(M-N)</b> <i>LncRNA-HIT</i> expression is detected in the digit perichondrial tissues, digit joint fields, and in the developing carpal/tarsal skeletal elements in E 13.5 forelimbs and hindlimbs. <b>(O)</b> Relative fold expression of <i>LncRNA-HIT</i> and <i>Hottip</i> expression in the E 11.5 limbs as determined by qRTPCR. Values represent the <i>Gapdh</i> normalized average expression of <i>LncRNA-HIT</i> and Hottip in the limb calculated from three independent analyses. Bars represent the standard deviation of the mean from the three independent assays. UGS = urogenital sinus, VB = vertebral body, GT = genital tubercle, GE = gut epithelium, SC = spinal cord.</p

siRNA-mediated reduction in <i>LncRNA-HIT</i> or <i>Snd1</i> results in reduced levels of 5’ HoxA gene expression.

<p><b>(A)</b> Relative gene expression after transfection with <i>LncRNA-HIT</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test. <b>(B)</b> Relative gene expression after transfection with <i>Snd1</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test.</p

Co-recruitment of <i>LncRNA-HIT</i> and p100 is required to stimulate gene expression from a synthetic locus.

<p><b>(A)</b> Analysis of UAS-luciferase reporter activation after GAL4-λN-BoxB<i>LncRNA-HIT</i> and GAL4-p100 recruitment. <b>(B)</b> Analysis of UAS-luciferase reporter activation in the absence of GAL4-p100. <b>(C)</b> Model of UAS-luciferase reporter activation in response to the recruitment of <i>LncRNA-HIT</i> and p100. For panels A and B, increasing dosages (0, 50, 100, 250, ng) of the BoxB or BoxB-<i>LncRNA-HIT</i> transcripts are represented by the black triangle.</p

Chondrogenic differentiation is impacted by siRNA-mediated reductions in Snd1 (p100) in micromass assays.

<p><b>(A)</b> Mesenchymal cell condensation is unaffected after transfection of the dissociated limb mesenchyme with a scrambled siRNA control. <b>(C and E)</b> After condensation, the scrambled control transfected limb mesenchyme are competent to differentiate into cartilage nodules that stain positively for the cartilage marker alcian blue. <b>(B,D,F)</b> Parallel assays transfecting the same limb mesenchyme with a <i>Snd1</i> siRNA cocktail severely impacts mesenchymal cell condensation producing few pre-cartilaginous condensations that can differentiate into mature cartilage nodules that stain positively with alcian blue. <b>(G)</b> Quantitation of the <i>Snd1</i> levels 24H after transfection with the <i>Snd1 siRNA cocktail</i> or scrambled siRNA controls reveal a seventy percent reduction in endogenous <i>Snd1</i> levels compared to parallel transfections using the scrambled siRNA controls. Values represent the average relative expression calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent assays. A Student’s t-test was used to determine significance of the averaged <i>Snd1</i> expression values compared to the scrambled <i>s</i>iRNA controls. A significant difference is indicated by an asterisk.</p