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  1. Article ; Online: Auxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation

    Judith Hyle / Mohamed Nadhir Djekidel / Justin Williams / Shaela Wright / Ying Shao / Beisi Xu / Chunliang Li

    Genome Biology, Vol 24, Iss 1, Pp 1-

    2023  Volume 30

    Abstract: Abstract Background CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription ... ...

    Abstract Abstract Background CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF. Results We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.
    Keywords CTCF ; Transcription ; Auxin-inducible degron ; CRISPR ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
    Subject code 570
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

    Mengli Zhang / Judith Hyle / Xiaowen Chen / Ye Xin / Yingcai Jin / Jianxiang Zhang / Xue Yang / Xinfeng Chen / Shaela Wright / Zhenling Liu / Wojciech Rosikiewicz / Beisi Xu / Liusheng He / Hong Liu / Nana Ping / Depei Wu / Feiqiu Wen / Chunliang Li / Peng Xu

    Genome Biology, Vol 25, Iss 1, Pp 1-

    2024  Volume 29

    Abstract: Abstract Background The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been ... ...

    Abstract Abstract Background The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated. Results Here, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription. Conclusions We identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment.
    Keywords CRISPR screen ; Genome editing ; RBM5 ; HOXA9 ; Acute myeloid leukemia ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
    Subject code 612 ; 570
    Language English
    Publishing date 2024-01-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Systematic characterization of the HOXA9 downstream targets in MLL-r leukemia by noncoding CRISPR screens

    Shaela Wright / Xujie Zhao / Wojciech Rosikiewicz / Shelby Mryncza / Judith Hyle / Wenjie Qi / Zhenling Liu / Siqi Yi / Yong Cheng / Beisi Xu / Chunliang Li

    Nature Communications, Vol 14, Iss 1, Pp 1-

    2023  Volume 15

    Abstract: Abstract Accumulating evidence indicates that HOXA9 dysregulation is necessary and sufficient for leukemic transformation and maintenance. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory ... ...

    Abstract Abstract Accumulating evidence indicates that HOXA9 dysregulation is necessary and sufficient for leukemic transformation and maintenance. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes. Here, we conduct dropout CRISPR screens against 229 HOXA9-bound peaks identified by ChIP-seq. Integrative data analysis identifies reproducible noncoding hits, including those located in the distal enhancer of FLT3 and intron of CDK6. The Cas9-editing and dCas9-KRAB silencing of the HOXA9-bound sites significantly reduce corresponding gene transcription and impair cell proliferation in vitro, and in vivo by transplantation into NSG female mice. In addition, RNA-seq, Q-PCR analysis, chromatin accessibility change, and chromatin conformation evaluation uncover the noncoding regulation mechanism of HOXA9 and its functional downstream genes. In summary, our work improves our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency.
    Keywords Science ; Q
    Subject code 570
    Language English
    Publishing date 2023-11-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Acute depletion of CTCF rewires genome-wide chromatin accessibility

    Beisi Xu / Hong Wang / Shaela Wright / Judith Hyle / Yang Zhang / Ying Shao / Mingming Niu / Yiping Fan / Wojciech Rosikiewicz / Mohamed Nadhir Djekidel / Junmin Peng / Rui Lu / Chunliang Li

    Genome Biology, Vol 22, Iss 1, Pp 1-

    2021  Volume 25

    Abstract: Abstract Background The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute ... ...

    Abstract Abstract Background The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription. Results Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription. Conclusions This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.
    Keywords ATAC-seq ; Auxin-induced degron ; Chromatin accessibility ; CTCF ; Transcription factor ; Proteomics ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
    Subject code 570
    Language English
    Publishing date 2021-08-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen

    Hao Zhang / Yang Zhang / Xinyue Zhou / Shaela Wright / Judith Hyle / Lianzhong Zhao / Jie An / Xujie Zhao / Ying Shao / Beisi Xu / Hyeong-Min Lee / Taosheng Chen / Yang Zhou / Xiang Chen / Rui Lu / Chunliang Li

    eLife, Vol

    2020  Volume 9

    Abstract: Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation ... ...

    Abstract Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. 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. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at 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.
    Keywords knock-in ; CRISPR screen ; HOXA9 ; transcriptional regulation ; mll-rearranged leukemia ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2020-10-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Inactivation of Ezh2 Upregulates Gfi1 and Drives Aggressive Myc-Driven Group 3 Medulloblastoma

    BaoHan T. Vo / Chunliang Li / Marc A. Morgan / Ilan Theurillat / David Finkelstein / Shaela Wright / Judith Hyle / Stephanie M.C. Smith / Yiping Fan / Yong-Dong Wang / Gang Wu / Brent A. Orr / Paul A. Northcott / Ali Shilatifard / Charles J. Sherr / Martine F. Roussel

    Cell Reports, Vol 18, Iss 12, Pp 2907-

    2017  Volume 2917

    Abstract: The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in ... ...

    Abstract The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor-promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.
    Keywords group 3 medulloblastoma ; MYC ; polycomb-repressive complex 2 ; PRC2 ; enhancer of zeste homology 2 ; EZH2 ; suppressor of zeste 12 homolog ; SUZ12 ; growth factor independent 1 ; GFI1 ; histone H3 modification ; Hox genes ; epigenetic repression ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2017-03-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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