Functional Interrogation ofHOXA9Regulome in MLLr Leukemia via Reporter-based CRISPR/Cas9 screen

Abstract

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, including human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL). HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies to treat HOXA9-driven leukemia. To mitigate these challenges, we generated the first HOXA9-mCherry knock-in reporter in an MLL-rearranged (MLLr) B-ALL cell line to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 mediated screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2 and its homolog USF1. USF1/USF2 depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of HOXA9-MEIS1 fusion protein rescued the impaired leukemia cell proliferation upon USF2 loss. Cut&Run analysis revealed the direct occupancy of USF2 onto HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

False-Positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5’ UTR annotations

“…Using FIMO (23) to search for transcription factor binding sites, we found the equivalent DNA sequence (CACGTG) matches the E-box motif recognized by bHLH transcription factors MYC/MAX (24) and USF1/USF2 (25,26). Recent work showed USF2 binds upstream of human Hoxa9, and that co-depletion of USF1 and USF2 decreases Hoxa9 expression in human tissue culture cells (27)(Figure 3A). Remarkably, this E-box appears to be universally conserved in vertebrates, with the only substitution being CACATG, which also functions as an E-box.…”

An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus

“…Loss of a single enhancer results in EZH2 enrichment at the INK4a/ARF promoters The INK4a/ARF locus is suppressed by loading of Polycomb on its promoters in stem cells and many tumors (Aguilo et al, 2011;Gamell et al, 2017;Li et al, 2009;Mosteiro et al, 2018;Yap et al, 2010;Zhang et al, 2019). Hence, we interrogated whether the promoter silencing upon enhancer deletions is due to the loading of EZH2 (polycomb complex subunit) on promoters.…”

“…Most studies carried out on INK4a/ARF transcriptional regulation have focused on promoter-driven mechanisms (Aloia et al, 2015;Hirosue et al, 2012;Lazorthes et al, 2015;Zhang et al, 2019). However, disease-associated variants identified in GWAS studies lie in the gene-desert region adjacent to CDKN2A/2B genes.…”

Reduced gene templates for supervised analysis of scale-limited CRISPR-Cas9 fitness screens

“…Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNAs (sgRNAs) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, part of the same pathway or genes coding for druggable proteins (Birsoy et al, 2015; Condon et al, 2021; Girardi et al, 2020; Parrish et al, 2021; Roesch et al, 2018; Słabicki et al, 2020; Su et al, 2020; Tarumoto et al, 2018; Turner and Turner,2021; Wheeler et al, 2020; Williams et al, 2020; Zhang et al, 2020; Zhu et al, 2021).…”

“…Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNAs (sgRNAs) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, part of the same pathway or genes coding for druggable proteins (Birsoy et al, 2015;Condon et al, 2021;Girardi et al, 2020;Parrish et al, 2021;Roesch et al, 2018;Słabicki et al, 2020;Su et al, 2020;Tarumoto et al, 2018;Turner and Turner, 2021;Wheeler et al, 2020;Williams et al, 2020;Zhang et al, 2020;Zhu et al, 2021). These scaled-limited screens are commonly used to test narrower biological hypotheses and they require an initial pooled population of cells of significantly smaller size compared to genome-wide screens, in order to obtain an appropriate library representation and multiplicity of infection following transfection and selection (Doench, 2018;Gonçalves et al, 2021;Miles et al, 2016;Peets et al, 2019).…”