FOX proteins are predicted to bind to putative <i>let-7a3/b</i> enhancer regions.

<p><b>(A)</b> The predicted existence of an upstream enhancer for the <i>let-7a3/b</i> locus was based on the epigenetic state at a region 10kb upstream of the TSS, outlined in green. In addition to being marked by H3K27Ac ChIP-Seq peaks with a localized dip in signal intensity, and peaks for the enhancer-associated histone acetyltransferase protein P300, this region showed dynamic changes in DNAse sensitivity. Note that a large DNAse sensitivity peak appears only in ES-derived NPCs, suggesting a differentiation state-specific chromatin opening at this region. At bottom, relative intensity of forkhead box protein ChIP-Seq from multiple cell types are pooled, with the darkest regions indicating intense FOX protein binding. Outlined in red are similar regions that show DNAse sensitivity beginning at the fetal brain stage that also colocalize with FOX protein binding. <b>(B)</b> A zoomed in view of the green region of increased DNAse sensitivity in PSC-derived NPCs. In blue are computationally predicted transcription factor binding sites from the ORCAtk database. The degree of genomic conservation along this region from the PhastCons64 database is shown in purple. At bottom are transcription factor ChIP-seq mapped peaks from the ENCODE database. The regions in green mark forkhead box transcription factor conserved motifs. Note that the forkhead box motifs co-localize with a region of highly conserved sequence, and the redundant binding of the forkhead box motif by many family members predicts that many such proteins can bind there.</p

Dynamically and constitutively transcribed <i>let-7</i> loci show distinct epigenetic signatures.

<p>Computationally imputed chromatin states generated by the ChromHMM algorithm at the same <i>let-7</i> loci. Each row represents one biological sample. These states show active transcriptional marks at the predicted TSS for <i>let-7a1/d/f1</i> in multiple cell types. At the <i>let-7a3/b</i> locus, ES cells, iPS cells, and PSC-derived NPCs have marks consistent with poised promoters, but later in differentiation active TSS marks appear at the same sites, reflecting changes in epigenetic state during neural differentiation. Epigenetic marks in K562 leukemia cells show active transcription at the RefSeq annotated <i>let-7a3/b</i> locus.</p

Transcription factors predicted to bind to the <i>let-7a3/b</i> promoter regulate primary <i>let-7a3/b</i> transcription.

<p><b>(A)</b> Comparison of transcription factors with experimentally determined binding sites to the <i>bona fide let-7a3/b</i> promoter from the ENCODE database with genes differentially expressed between tissue-derived NPCs (in which <i>let-7a3/b</i> is abundantly transcribed) and PSC-derived NPCs (in which it is not). 10 genes were present in both sets, and are shown at right, ranked by their fold change of expression between tissue-derived and PSC-derived NPCs from microarray based gene expression measurements. We knocked down several of these candidate <i>let-7</i> regulator transcription factors in tissue-derived NPCs. <b>(B)</b> Knockdown of the <i>TFAP2C</i> gene encoding the AP-2γ protein, and of the <i>MYCN</i> gene increase transcription of several <i>let-7</i> genes. Data shown are representative of 3 independent experiments. <b>(C)</b> Knockdown of <i>EGR1</i> increases transcription of primary <i>let-7b</i> and other <i>let-7</i> genes. Error bars are ± SEM from n = 3 biological replicates.</p

A role for FOX proteins in regulation of pri-let-7s.

<p><b>(A)</b> By filtering for FOX genes that are actively transcribed in our neural cells and differentially expressed between PSC-derived NPCs and Tissue-derived NPCs, we generated a list of candidate proteins that might mediate changes in <i>let-7a3/b</i> transcription. <b>(B)</b> Knockdown of FOX proteins, FOXP2, FOXP4 and FOXN3 in Tissue-derived NPCs show distinct effects on primary <i>let-7</i> transcript levels. Statistics were performed across three independent experiments (two-tailed t-test, p < 0.05).</p

Dynamic transcriptional regulation of some <i>pri-let-7</i> transcripts.

<p>Chromatin-associated RNA-seq reads were mapped onto two distinct polycistronic <i>let-7</i> loci. At left, the <i>let-7a3/b</i> locus, is dynamic, while at right, the <i>let-7a1/d/f1</i> locus, is constitutively expressed. Reads are shown for ESC, iPSC, PSC-derived NPC, and neural tissue-derived NPC stages. These reads are aligned with validated primary miRNA transcripts from RACE PCR experiments in green and RefSeq annotated genes in blue²⁵. Note that Chromatin-associated RNA-seq and RACE PCR annotated transcripts demonstrate the existence of longer transcripts from different transcriptional start sites than suggested by the RefSeq annotation. In the case of <i>let-7a1/d/f1</i>, this discrepancy extends to the strand from which initial transcription occurs.</p

Expression of <i>pri-let-7</i> during neural specification.

<p>Pluripotent stem cells were differentiated through the neural lineage to neural progenitor cells (NPCs) and then to neurons. Using RT-PCR with primers specific to the <i>let-7</i> miRNAs at different stages of processing, we tested changes in expression of the <i>pri-let-7</i>s <b>(A)</b> and their mature forms <b>(B)</b>. While all mature miRNAs increased over the course of differentiation, only a subset (marked with dotted lines), the dynamically regulated <i>let-7</i>s, also increased before processing, at the primary <i>let-7</i> stage. RT-PCRs were also performed beginning with ES cells. <b>(C)</b> Graphic comparing the length of the RefSeq annotated <i>let-7a3/b</i> with our predicted transcript. Stars mark primer pairs for RT-PCR along the full transcript. <b>(D)</b> RT-PCR of <i>pri-let-7a3/b</i> transcript in tissue-derived NPCs, in which transcription is abundant. In control, siDGCR8 (to block Microprocessor function and pri-to-pre conversion), and siDICER (to block pre-to-mature conversion) conditions. When Microprocessor is disabled, the entire <i>let-7a3/b</i> transcript accumulates.</p

Data from Roadmap project in GEO database.

<p>Data from Roadmap project in GEO database.</p

Defining Transcriptional Regulatory Mechanisms for Primary let-7 miRNAs

<div><p>The <i>let-7</i> family of miRNAs have been shown to control developmental timing in organisms from <i>C</i>. <i>elegans</i> to humans; their function in several essential cell processes throughout development is also well conserved. Numerous studies have defined several steps of post-transcriptional regulation of <i>let-7</i> production; from pri-miRNA through pre-miRNA, to the mature miRNA that targets endogenous mRNAs for degradation or translational inhibition. Less-well defined are modes of transcriptional regulation of the pri-miRNAs for <i>let-7</i>. <i>let-7</i> pri-miRNAs are expressed in polycistronic fashion, in long transcripts newly annotated based on chromatin-associated RNA-sequencing. Upon differentiation, we found that some <i>let-7</i> pri-miRNAs are regulated at the transcriptional level, while others appear to be constitutively transcribed. Using the Epigenetic Roadmap database, we further annotated regulatory elements of each polycistron identified putative promoters and enhancers. Probing these regulatory elements for transcription factor binding sites identified factors that regulate transcription of <i>let-7</i> in both promoter and enhancer regions, and identified novel regulatory mechanisms for this important class of miRNAs.</p></div

Data from ENCODE project in GEO database (or DCC Accession #).

<p>Data from ENCODE project in GEO database (or DCC Accession #).</p