Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells

Abstract

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

Figure 1. High-resolution genome-wide mapping of core ES cell transcription factors with ChIP-seq

A. Summary of binding data for Oct4, Sox2, Nanog and Tcf3. 14,230 sites are co-bound genome wide and mapped to either promoter proximal (TSS +/− 8kb, dark green, 27% of binding sites), genic (>8kb from TSS, middle green, 30% of binding sites), or intergenic (light green, 43% of binding sites). The promoter proximal binding sites are associated with 3,289 genes. B. (upper) Binding of Oct4 (blue), Sox2 (purple), Nanog (orange) and Tcf3 (red) across 37.5kb of mouse chromosome 3 surrounding the Sox2 gene (black box below the graph, arrow indicates transcription start site). Short sequences uniquely and perfectly mapping to the genome were extended to 200bp (maximum fragment length) and scored in 25bp bins. The score of the bins were then normalized to the total number of reads mapped. Oct4/Sox2 DNA binding motifs (Loh et al., 2006) were mapped across the genome and are shown as grey boxes below the graph. Height of the box reflects the quality of the motif. (lower) Detailed analysis of three enriched regions (Chromosome 3: 4,837,600-34,838,300, 34,845,300-34,846,000, and 34,859,900–34,860,500) at the Sox2 gene indicated with dotted boxes above. The 5’ most base from ChIP-seq were separated by strand and binned into 25bp regions. Sense (darker tone) and anti-sense (light tone) of each of the four factors tested are directed towards the binding site, which in each case occurs at a high-confidence Oct/Sox2 DNA binding motif indicated below.

Figure 2. Identification of miRNA promoters

A. Description of algorithm for miRNA promoter identification. A library of candidate transcriptional start sites was generated with histone H3 lysine 4 tri-methyl (H3K4me3) location analysis data from multiple tissues (Barski et al., 2007; Guenther et al., 2007; Mikkelsen et al., 2007). Candidates were scored to assess likelihood that they represent true miRNA promoters. Based on scores, a list of mouse and human miRNA promoters was assembled. Additional details can be found in Supplemental Text. B. Examples of identified miRNA promoter regions. A map of H3K4me3 enrichment is displayed in regions neighbouring selected human and mouse miRNAs for multiple cell types: human ES cells (hES), REH human pro-B cell line (B cell), primary human hepatocytes (Liver), primary human T cells (T cell), mouse ES cells (mES), neural precursor cells (NPCs) and mouse embryonic fibroblasts (MEFs). miRNA promoter coordinates were confirmed by distance to mature miRNA genomic sequence, conservation and EST data (shown as solid line where available). Predicted transcriptional start site and direction of transcription are noted by an arrow, with mature miRNA sequences indicated (red). CpG islands, commonly found at promoters, are indicated (green). Dotted lines denote presumed transcripts. C. Confirmation of predicted transcription start sites for active miRNAs using chromatin modifications. Normalized ChIP-seq counts for H3K4me3 (red), H3K79me2 (blue) and H3K36me3 (green) are shown for two miRNA genes where EST data was unavailable. Predicted start site (arrow), CpG islands (green bar), presumed transcript (dotted lines) and miRNA positions (red bar) are shown. D. Most human and mouse miRNA promoters show evidence of H3K4me3 enrichment in multiple tissues.

Figure 3. Oct4, Sox2, Nanog and Tcf3 occupancy and regulation of miRNA promoters

A. Oct4 (blue), Sox2 (purple), Nanog (orange) and Tcf3 (red) binding is shown at four murine miRNA genes as in Figure 1A. H3K4me3 enrichment in ES cells is indicated by shading across genomic region. Presumed transcripts are shown as dotted lines. Coordinates for the mmu-mir-290-295 cluster are derived from NCBI build 37. B. Oct4 ChIP enrichment ratios (ChIP-enriched versus total genomic DNA) are shown across human miRNA promoter region for the hsa-mir-302 cluster. H3K4me3 enrichment in ES cells is indicated by shading across genomic region. C. Schematic of miRNAs with conserved binding by the core transcription factors in ES cells. Transcription factors are represented by dark blue circles and miRNAs are represented by purple hexagons. D. Quantitative RT-PCR analysis of RNA extracted from ZHBTc4 cells in the presence or absence of doxycycline treatment. Fold change was calculated for each pri-miRNA for samples from 12 hours and 24 hours of doxycyline treatment relative to those from untreated cells. Transcript levels were normalized to Gapdh levels. Error bars indicate standard deviation derived from triplicate PCR reactions.

Figure 4. Regulation of Oct4/Sox2/Nanog/TCF3-bound miRNAs during differentiation

A. Pie charts showing relative contributions of miRNAs to the complete population of miRNAs in mES cells (red), MEFs (blue) and neural precursors (NPCs, green) based on quantification of miRNAs from by small RNA sequencing. A full list of the miRNAs identified can be found in Table S9. B. Normalized frequency of detection of individual mature miRNAs whose promoters are occupied by Oct4/Sox2/Nanog/ Tcf3 in mouse. Red line in center and right panel show the level of detection in ES cells. C. Histogram of changes in frequency of detection. Changes for miRNAs whose promoters are occupied by Oct4, Sox2, Nanog and Tcf3 in mouse are shown as bars (red for ES enriched, blue for MEF enriched and green for NPC enriched). The background frequency for non-occupied miRNAs is shown as a grey line.

Figure 5. Polycomb represses lineage-specific miRNAs in ES cells

A. Suz12 (light green) and H3K27me3 (dark green, Mikkelsen et al., 2007) binding are shown for two miRNA genes in murine ES cells. Predicted start sites (arrow), CpG islands (green bar), presumed miRNA primary transcript (dotted line) and mature miRNA (red bar) are shown. B. Expression analysis of miRNAs from mES cells based on quantitative small RNA sequencing. Cumulative distributions for Polycomb-bound miRNAs (green line) and all miRNAs (grey line) are shown. C. Expression analysis of miRNAs occupied by Suz12 in mES cells. Relative counts are shown for mES (red), NPCs (orange) and MEFs (yellow). H3K27me3 (green line) and H3K4me3 (blue line) mapped reads are shown for mES cells, MEFs and NPCs (Mikkelsen et al., 2007). D. Schematic of a subset of miRNA genes occupied by Suz12 in both mES and hES cells as in Figure 3C. miRNA genes where Oct4/Sox2/Nanog/Tcf3 are also present are indicated. Cells known to selectively express these miRNAs based on computation predictions (Farh et al., 2005) or experimental confirmation (Landgraf et al., 2007) are indicated. The Polycomb group (PcG) protein Suz12 is represented by a green circle.

Figure 6. miRNA modulation of the gene regulatory network in ES cells

A. An incoherent feed-forward motif (Alon, 2007) involving a miRNA repression of a transcription factor target gene is illustrated (left). Transcription factors are represented by dark blue circles, miRNAs in purple hexagons, protein-coding gene in pink rectangles and proteins in orange ovals. Selected instances of this network motif identified in ES cells based on data from Sinkkonen et al., 2008 or data in Figure S11 are shown (right). B. Second model of incoherent feed-forward motif (Alon 2007) involving protein repression of a miRNA is illustrated (left). In ES cells, Lin28 blocks the maturation of primary Let-7g (Visiwanthan et al., 2008). Lin28 and the Let-7g gene are occupied by Oct4/Sox2/Nanog/Tcf3. Targetscan prediction (Grimson et al., 2007), of Lin28 by mature Let-7g is noted (purple dashed line, right). C. A coherent feed-forward motif (Alon 2007) involving miRNA repression of a transcriptional repressor that regulates a transcription factor target gene is illustrated (left). This motif is found in ES cells, where mir-290-295 miRNAs repress Rbl2 indirectly maintaining the expression of Dnmt3a and Dnmt3a, which are also occupied at their promoters by Oct4/Sox2/Nanog/Tcf3 (right).

Figure 7. Multi-level regulatory network controlling ES cell identity

Updated map of ES cell regulatory circuitry is shown. Interconnected auto-regulatory loop is shown to the left. Active genes are shown at the top right, and inactive genes are shown at the bottom right. Transcription factors are represented by dark blue circles, and Suz12 by a green circle. Gene promoters are represented by red rectangles, gene products by orange circles, and miRNA promoters are represented by purple hexagons.