Biology subjects

Harman, J. R.

Publications and source records attributed to Harman, J. R..

4 recordsLinked to original sources

bioRxiv2025

Sustained MYB activity drives emergent enhancer activation and precise enhancer-promoter interactions

Transcription factors (TFs) are key effectors of enhancer activity. MYB is a critical hematopoietic TF that is frequently dysregulated in cancer. Despite its well-established role, the exact mechanisms by which MYB influences enhancer function--and the specific stages of enhancer activation at which it operates--remain poorly understood. Using high resolution Micro-Capture-C, we show that upon MYB degradation, highly defined enhancer-promoter interactions at specific MYB binding sites are lost. Loss of these interactions, together with other hallmarks of enhancer activity--reduced H3 lysine-27 acetylation and enhancer RNA transcription--correlates with significant downregulation of target gene expression in leukemia, indicating that MYB mediates transcription activation via maintenance of enhancer function. When anchored to DNA within a gene desert region that is devoid of histone marks and active transcription, the MYB transactivation domain is sufficient and necessary for the nucleation of an enhancer-like region. This results in the activation of transcription from distal cryptic elements and the establishment of long-range chromatin interactions up to 400 kb away from the anchor point. Together, these results indicate that MYB activity alone is sufficient to induce long-range interactions and transcription, achieving this through highly precise enhancer-promoter crosstalk.

genomics↗

bioRxiv2024

The fetal specific gene LIN28B is essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. First, by comparing transcriptomes of human fetal HSPC to adult HSPC we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stemness, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilised a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this model, LIN28B-expressing leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Finally, In the absence of LIN28B, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in fetal cells in the absence of co-operating mutations. It has a role in making leukemias more aggressive, suggesting it is a potential target in LIN28B-expressing leukemias.

cancer biology↗

bioRxiv2021

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.

developmental biology↗

bioRxiv2020

Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than studying single TF binding events in isolation. Chromosomal translocations of the Mixed Lineage Leukemia gene (MLL) produce MLL fusion proteins such as MLL-AF4, causing poor prognosis acute lymphoblastic leukemias (ALLs). MLL-AF4 is thought to drive leukemogenesis by directly binding to genes and inducing aberrant overexpression of key gene targets, including anti-apoptotic factors such as BCL-2. However, this model minimizes the potential for circuit generated regulatory outputs, including gene repression. To better understand the MLL-AF4 driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors, including RUNX1, that regulate target genes using feed-forward loop and cascade motifs. We used CRISPR screening in the presence of the BCL-2 inhibitor venetoclax to identify functional impacts on apoptosis. This identified an MLL-AF4:RUNX1 cascade that represses CASP9, perturbation of which disrupts venetoclax induced apoptosis. This illustrates how our GRN can be used to better understand potential mechanisms of drug resistance acquisition. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/179796v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@cca8f2org.highwire.dtl.DTLVardef@67b289org.highwire.dtl.DTLVardef@1fa44aeorg.highwire.dtl.DTLVardef@1b32487_HPS_FORMAT_FIGEXP M_FIG A network model of the MLL-AF4 regulatory landscape identifies feed-forward loop and cascade motifs. Functional screening using CRISPR and venetoclax identified an MLL-AF4:RUNX1:CASP9 repressive cascade that impairs drug-induced cell death. C_FIG

genomics↗