Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells

Abstract

The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells

“…Hox genes play highly conserved roles in modulating regional identity and programs of cellular differentiation in a temporally and spatially restricted manner (21). During RA-induced differentiation of murine ES cells, Hoxa1 is a direct target of RA signaling and is one of the most rapidly induced genes through mechanisms involving control of elongation of paused Pol II (RNA polymerase II) (22)(23)(24). This finding is consistent with it being an important early determinant in ES cell differentiation.…”

“…The advantage of this approach is that it permits the quantification of both total steady-state levels of RNA and nascent transcriptional activity per cell. We find that the total steady-state levels of Nanog remain relatively stable over the early time course of RA-induced differentiation as measured by RNA-seq, quantitative PCR, and FISH (22). In contrast, there is a progressive decline in the levels of nascent transcripts from 2 h onward, with fewer transcripts per cell and fewer cells with nascent transcripts (Fig.…”

“…S1C). The striking shift in binding-site distribution of Hoxa1 correlates with temporal changes in expression of markers for cell lineage and differentiation processes that reveal different cellular states exist at 2 and 12 h (22). Interestingly, a recent study on single-cell transcriptome analysis of mouse ES cells differentiated with RA over a 96-h time course also showed similar progressive changes in differentiation and that 12 h was an important transition point for exit from pluripotency (39).…”

“…During RA-induced neuro-ectodermal differentiation of murine ES cells, Hoxa1 is one of the most rapidly induced genes (22,24). In this RA-induced differentiation paradigm, monitoring events at the level of single cells using methods for single-molecule RNA FISH, we found a robust and relatively uniform RA response by the most rapidly induced genes, including Hoxa1 (22). Following initial activation of Hoxa1, we investigated its early downstream target genes after 2 and 12 h of RA-induced differentiation.…”

“…S1), but this cohort includes the core pluripotency genes Nanog and Sox2. In light of the relatively homogenous early differentiation of the ES cells (22), these shifts in binding distribution likely reflect differences in the progressive differentiation states between 2 and 12 h, rather than heterogeneity in the population.…”