Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells.

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of ß-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.

Exploiting genetic variation to uncover rules of transcription factor binding and chromatin accessibility.

Single-nucleotide variants that underlie phenotypic variation can affect chromatin occupancy of transcription factors (TFs). To delineate determinants of in vivo TF binding and chromatin accessibility, we introduce an approach that compares ChIP-seq and DNase-seq data sets from genetically divergent murine erythroid cell lines. The impact of discriminatory single-nucleotide variants on TF ChIP signal enables definition at single base resolution of in vivo binding characteristics of nuclear factors GATA1, TAL1, and CTCF. We further develop a facile complementary approach to more deeply test the requirements of critical nucleotide positions for TF binding by combining CRISPR-Cas9-mediated mutagenesis with ChIP and targeted deep sequencing. Finally, we extend our analytical pipeline to identify nearby contextual DNA elements that modulate chromatin binding by these three TFs, and to define sequences that impact kb-scale chromatin accessibility. Combined, our approaches reveal insights into the genetic basis of TF occupancy and their interplay with chromatin features.

A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition.

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.