MEIS inhibitors reduce the viability of primary leukemia cells and Stem cells by inducing apoptosis.

Leukemia stem cells (LSCs) exhibit self-renewal, resistance to standard treatments, and involvement in leukemia relapse. Higher Myeloid Ecotropic Integration Site-1 (MEIS1) expression in leukemic blast samples has been linked to resistance to conventional treatment. We studied the MEIS1 and associated factors in relapsed LSCs and assessed the effect of recently developed MEIS inhibitors (MEISi). Meis1 gene expression was found to be higher in patients with leukemia and relapsed samples. The majority of CD123+ and CD34+ LSCs demonstrated higher MEIS1/2/3 content. Depending on the patient chemotherapy regimen, Meis1 expression increased in relapsed samples. Although there are increased Meis2, Meis3, Hoxa9, Pbx1, or CD34 expressions in the relapsed patients, they are not correlated with Meis1 content in every patient or regimen. MEISi has reduced MEIS1 transcriptional activity and LSC cell survival by apoptosis. Pharmacological targeting with MEISi in LSCs could have a potential effect in limiting leukemia relapse and chemotherapeutic resistance.

Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors.

ABSTRACT: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

Transcriptional control of leukemogenesis by the chromatin reader SGF29.

ABSTRACT: Aberrant expression of stem cell-associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated "stemness" network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)-associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain-containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.

Meis1 establishes the pre-hemogenic endothelial state prior to Runx1 expression.

Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

Newly developed MEIS inhibitor selectively blocks MEISHigh prostate cancer growth and induces apoptosis.

Prostate cancer (PCa) is the second most diagnosed cancer in males. Understanding the molecular mechanism and investigation of novel ways to block PCa growth or metastasis are vital and a medical necessity. In this study, we examined differential expression of MEIS1/2/3 and its associated factors in PCa cell lines. MEIS1/2/3 content, reactive oxygen species, and cell cycle status were analyzed in PCa cells post MEIS inhibitor (MEISi) treatments, which is developed in our laboratory as a first-in-class small molecule inhibitor. A correlation was detected between MEIS content and MEISi IC50 values of PCa cells. MEISi decreased the viability of PC-3, DU145, 22Rv-1 and LNCaP cells, and significantly increased apoptosis in parallel with the increased cellular ROS content. The efficacy of MEISi was shown to positively correlate with the levels of MEIS1/2/3 proteins and the long term exposure to MEISi elevated MEIS1/2/3 protein content in PCa cells. Our findings suggest that MEISi could be used to target PCa with high MEIS expression in order to reduce PCa viability and growth; however, more research is needed before this can be translated into clinical settings.

MEIS1 Is a Common Transcription Repressor of the miR-23a and NORHA Axis in Granulosa Cells.

MicroRNA-23a (miR-23a) is an endogenous small activating RNA (saRNA) involved in ovarian granulosa cell (GC) apoptosis and sow fertility by activating lncRNA NORHA transcription. Here, we reported that both miR-23a and NORHA were repressed by a common transcription factor MEIS1, which forms a small network regulating sow GC apoptosis. We characterized the pig miR-23a core promoter, and the putative binding sites of 26 common transcription factors were detected in the core promoters of both miR-23a and NORHA. Of them, transcription factor MEIS1 expression was the highest in the ovary, and widely distributed in various ovarian cells, including GCs. Functionally, MEIS1 is involved in follicular atresia by inhibiting GC apoptosis. Luciferase reporter and ChIP assays showed that transcription factor MEIS1 represses the transcription activity of miR-23a and NORHA through direct binding to their core promoters. Furthermore, MEIS1 represses miR-23a and NORHA expression in GCs. Additionally, MEIS1 inhibits the expression of FoxO1, a downstream of the miR-23a/NORHA axis, and GC apoptosis by repressing the miR-23a/NORHA axis. Overall, our findings point to MEIS1 as a common transcription repressor of miR-23a and NORHA, and develop the miR-23a/NORHA axis into a small regulatory network regulating GC apoptosis and female fertility.

Aggressive High-grade Uterine Sarcoma Harboring MEIS1-NCOA2 Fusion and Amplification of Multiple 12q13-15 Genes: A Case Report With Morphologic, Immunohistochemical, and Molecular Analysis.

MEIS1-NCOA1/2 fusions are recently described gene rearrangements found in rare sarcomas, mainly involving the genitourinary and gynecologic tracts, with 3 cases reported in the uterine corpus. Although local recurrence was very common, no death has been reported, and some investigators consider these sarcomas low grade. Amplification of genes located at the 12q13-15 locus, especially MDM2 , is the hallmark genetic abnormality in well-differentiated and dedifferentiated liposarcoma of the soft tissue. Some uterine tumors have also been reported to harbor MDM2 amplification, including a proportion of Müllerian adenosarcomas, BCOR fusion-positive high-grade endometrial stromal sarcoma, BCORL1 -altered high-grade endometrial stromal sarcoma, rare JAZF1 fusion-positive low-grade endometrial stromal sarcoma, rare undifferentiated uterine sarcoma, and a single case of MEIS1-NCOA2 fusion sarcoma. Here, we report a case of high-grade MEIS1-NCOA2 fusion uterine sarcoma which also harbored amplification of multiple 12q13-15 genes, including MDM2 , CDK4 , MDM4 , and FRS2 , that exhibited aggressive clinical course leading to patient's death within 2 yr of the initial diagnosis. To the best of our knowledge, this is the first documented case of fatal MEIS1-NCOA2 fusion uterine sarcoma, and the second case of MEIS1-NCOA2 fusion uterine sarcoma that also harbors MDM2 amplification.

Npm1 haploinsufficiency in collaboration with MEIS1 is sufficient to induce AML in mice.

NPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML). Mutations in the NPM1 gene result in the increased export of NPM1 to the cytoplasm (NPM1c) and are associated with multiple transforming events including the aberrant upregulation of MEIS1 that maintains stem cell and cell cycle-associated pathways in NPM1c AML. However, another consequence of the NPM1c mutation is the inadequate levels of NPM1 wild-type in the nucleus and nucleolus, caused by the loss of one wild-type allele in addition to enforced NPM1 nuclear export. The contribution of NPM1 haploinsufficiency independently of the NPM1 mutation to AML development and its relationship with MEIS1 function is poorly understood. Using mouse models, our study shows that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce a fully penetrant AML in mice that transcriptionally resembles human NPM1c AML. NPM1 haploinsufficiency alters MEIS1-binding occupancies such that it binds the promoter of the oncogene structural maintenance of chromosome protein 4 (SMC4) in NPM1 haploinsufficient AML cells but not in NPM1 wild-type-harboring Hoxa9/Meis1-transformed cells. SMC4 is higher expressed in haploinsufficient and NPM1c+ AML cells, which are more vulnerable to the disruption of the MEIS1-SMC4 axis compared with AML cells with nonmutated NPM1. Taken together, our study underlines that NPM1 haploinsufficiency on its own is a key factor of myeloid leukemogenesis and characterizes the MEIS1-SMC4 axis as a potential therapeutic target in this AML subtype.

A pro B cell population forms the apex of the leukemic hierarchy in Hoxa9/Meis1-dependent AML.

Relapse is a major challenge to therapeutic success in acute myeloid leukemia (AML) and can be partly associated with heterogeneous leukemic stem cell (LSC) properties. In the murine Hoxa9/Meis1-dependent (H9M) AML model, LSC potential lies in three defined immunophenotypes, including Lin-cKit+ progenitor cells (Lin-), Gr1+CD11b+cKit+ myeloid cells, and lymphoid cells (Lym+). Previous reports demonstrated their interconversion and distinct drug sensitivities. In contrast, we here show that H9M AML is hierarchically organized. We, therefore, tracked the developmental potential of LSC phenotypes. This unexpectedly revealed a substantial fraction of Lin- LSCs that failed to regenerate Lym+ LSCs, and that harbored reduced leukemogenic potential. However, Lin- LSCs capable of producing Lym+ LSCs as well as Lym+ LSCs triggered rapid disease development suggestive of their high relapse-driving potential. Transcriptional analyses revealed that B lymphoid master regulators, including Sox4 and Bach2, correlated with Lym+ LSC development and presumably aggressive disease. Lentiviral overexpression of Sox4 and Bach2 induced dedifferentiation of H9M cells towards a lineage-negative state in vitro as the first step of lineage conversion. This work suggests that the potency to initiate a partial B lymphoid primed transcriptional program as present in infant AML correlates with aggressive disease and governs the H9M LSC hierarchy.

Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

Therapeutic targeting in pediatric acute myeloid leukemia with aberrant HOX/MEIS1 expression.

Despite advances in the clinical management of childhood acute myeloid leukemia (AML) during the last decades, outcome remains fatal in approximately one third of patients. Primary chemoresistance, relapse and acute and long-term toxicities to conventional myelosuppressive therapies still constitute significant challenges and emphasize the unmet need for effective targeted therapies. Years of scientific efforts have translated into extensive insights on the heterogeneous spectrum of genetics and oncogenic signaling pathways of AML and identified a subset of patients characterized by upregulation of HOXA and HOXB homeobox genes and myeloid ecotropic virus insertion site 1 (MEIS1). Aberrant HOXA/MEIS1 expression is associated with genotypes such as rearrangements in Histone-lysine N-methyltransferase 2A (KMT2A-r), nucleoporin 98 (NUP98-r) and mutated nucleophosmin (NPM1c) that are found in approximately one third of children with AML. AML with upregulated HOXA/MEIS1 shares a number of molecular vulnerabilities amenable to recently developed molecules targeting the assembly of protein complexes or transcriptional regulators. The interaction between the nuclear scaffold protein menin and KMT2A has gained particular interest and constitutes a molecular dependency for maintenance of the HOXA/MEIS1 transcription program. Menin inhibitors disrupt the menin-KMT2A complex in preclinical models of KMT2A-r, NUP98-r and NPM1c acute leukemias and its occupancy at target genes leading to leukemic cell differentiation and apoptosis. Early-phase clinical trials are either ongoing or in development and preliminary data suggests tolerable toxicities and encouraging efficacy of menin inhibitors in adults with relapsed or refractory KMT2A-r and NPM1c AML. The Pediatric Acute Leukemia/European Pediatric Acute Leukemia (PedAL/EUPAL) project is focused to advance and coordinate informative clinical trials with new agents and constitute an ideal framework for testing of menin inhibitors in pediatric study populations. Menin inhibitors in combination with standard chemotherapy or other targeting agents may enhance anti-leukemic effects and constitute rational treatment strategies for select genotypes of childhood AML, and provide enhanced safety to avoid differentiation syndrome. In this review, we discuss the pathophysiological mechanisms in KMT2A-r, NUP98-r and NPM1c AML, emerging molecules targeting the HOXA/MEIS1 transcription program with menin inhibitors as the most prominent examples and future therapeutic implications of these agents in childhood AML.

Normal and Aberrant TALE-Class Homeobox Gene Activities in Pro-B-Cells and B-Cell Precursor Acute Lymphoblastic Leukemia.

Homeobox genes encode transcription factors regulating basic developmental processes. They are arranged according to sequence similarities of their conserved homeobox in 11 classes, including TALE. Recently, we have reported the so-called TALE-code. This gene signature describes physiological expression patterns of all active TALE-class homeobox genes in the course of hematopoiesis. The TALE-code allows the evaluation of deregulated TALE homeobox genes in leukemia/lymphoma. Here, we extended the TALE-code to include the stages of pro-B-cells and pre-B-cells in early B-cell development. Detailed analysis of the complete lineage of B-cell differentiation revealed expression of TALE homeobox genes IRX1 and MEIS1 exclusively in pro-B-cells. Furthermore, we identified aberrant expression of IRX2, IRX3 and MEIS1 in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) which originates from early B-cell progenitors. The data showed correlated activities of deregulated TALE-class members with particular BCP-ALL subtype markers, namely IRX2 with TCF3/E2A-fusions, IRX3 with ETV6/TEL-fusions, and MEIS1 with KMT2A/MLL-fusions. These correlations were also detected in BCP-ALL cell lines which served as experimental models. We performed siRNA-mediated knockdown experiments and reporter gene assays to analyze regulatory connections. The results showed mutual activation of IRX1 and TCF3. In contrast, IRX2 directly repressed wild-type TCF3 while the fusion gene TCF3::PBX1 lost the binding site for IRX2 and remained unaltered. IRX3 mutually activated fusion gene ETV6::RUNX1 while activating itself by aberrantly expressed transcription factor KLF15. Finally, KMT2A activated MEIS1 which in turn supported the expression of IRX3. In summary, we revealed normal TALE homeobox gene expression in early B-cell development and identified aberrant activities of IRX2, IRX3 and MEIS1 in particular subtypes of BCP-ALL. Thus, these TALE homeobox genes may serve as novel diagnostic markers and therapeutic targets.

Hoxa9/meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance.

HOXA9 and MEIS1 are co-expressed in over 50% of acute myeloid leukaemia (AML) and play essential roles in leukaemogenesis, but the mechanisms involved are poorly understood. Diverse animal models offer valuable tools to recapitulate different aspects of AML and link in vitro studies to clinical trials. We generated a double transgenic zebrafish that enables hoxa9 overexpression in blood cells under the draculin (drl) regulatory element and an inducible expression of meis1 through a heat shock promoter. After induction, Tg(drl:hoxa9;hsp70:meis1) embryos developed a preleukaemic state with reduced myeloid and erythroid differentiation coupled with the poor production of haematopoietic stem cells and myeloid progenitors. Importantly, most adult Tg(drl:hoxa9;hsp70:meis1) fish at 3 months old showed abundant accumulations of immature myeloid precursors, interrupted differentiation and anaemia in the kidney marrow, and infiltration of myeloid precursors in peripheral blood, resembling human AML. Genome-wide transcriptional analysis also confirmed AML transformation by the transgene. Moreover, the dihydroorotate dehydrogenase (DHODH) inhibitor that reduces leukaemogenesis in mammals effectively restored haematopoiesis in Tg(drl:hoxa9;hsp70:meis1) embryos and improved their late survival. Thus, Tg(drl:hoxa9;hsp70:meis1) zebrafish is a rapid-onset high-penetrance AML-like disease model, which provides a novel tool to harness the unique advantages of zebrafish for mechanistic studies and drug screening against HOXA9/MEIS1 overexpressed high-risk AML.

Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice.

INTRODUCTION: The principal voltage-gated Na+ channel, NaV1.5 governs heart excitability and conduction. NaV1.5 dysregulation is responsible for ventricular arrhythmias and subsequent sudden cardiac death (SCD) in post-infarct hearts. The transcription factor Meis1 performs a significant role in determining differentiation fate and regenerative capability of cardiomyocytes. However, the functions of Meis1 in ischemic arrhythmias following myocardial infarction (MI) are still largely undefined. OBJECTIVES: Here we aimed to study whether Meis1 could act as a key regulator to mediate cardiac Na+ channel and its underlying mechanisms. METHODS: Heart-specific Meis1 overexpression was established by AAV9 virus injection in C57BL/6 mice. The QRS duration, the incidence of ventricular arrhythmias and cardiac conduction velocity were evaluated by ECG, programmed electrical stimulation and optical mapping techniques respectively. The conventional patch clamp technique was performed to explore the INa characteristics of isolated mouse ventricular myocytes. In vitro, Meis1 was also overexpressed in hypoxic-treated neonatal cardiomyocytes. The analysis of immunoblotting and immunofluorescence were used to detect the changes in the expression of NaV1.5 in each group. RESULTS: We found that forced expression of Meis1 rescued the prolongation of QRS complex, produced anti-arrhythmic activity and improved epicardial conduction velocity in infarcted mouse hearts. In terms of mechanisms, cardiac electrophysiological changes of MI mice can be ameliorated by the recovery of Meis1, which is characterized by the restoration of INa current density and NaV1.5 expression level of cardiomyocytes in the marginal zone of MI mouse hearts. Furthermore, in vitro studies showed that Meis1 was also able to rescue hypoxia-induced decreased expression and dysfunction of NaV1.5 in ventricular myocytes. We further revealed that E3 ubiquitin ligase CDC20 led to the ubiquitination and degradation of Meis1, which blocked the transcriptional regulation of SCN5A by Meis1 and ultimately led to the electrophysiological remodeling in ischemic-hypoxic cardiomyocytes. CONCLUSION: CDC20 mediates ubiquitination of Meis1 to govern the transcription of SCN5A and cardiac electrical conduction in mouse cardiomyocytes. This finding uncovers a new mechanism of NaV1.5 dysregulation in infarcted heart, and provides new therapeutic strategies for malignant arrhythmias and sudden cardiac death following MI.

In vivo genome-wide CRISPR screening in murine acute myeloid leukemia uncovers microenvironmental dependencies.

Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the ß-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.

3D Chromatin Organization Involving MEIS1 Factor in the cis-Regulatory Landscape of GJB2.

The human genome is covered by 8% of candidate cis-regulatory elements. The identification of distal acting regulatory elements and an understanding of their action are crucial to determining their key role in gene expression. Disruptions of such regulatory elements and/or chromatin conformation are likely to play a critical role in human genetic diseases. Non-syndromic hearing loss (i.e., DFNB1) is mostly due to GJB2 (Gap Junction Beta 2) variations and DFNB1 large deletions. Although several GJB2 cis-regulatory elements (CREs) have been described, GJB2 gene regulation remains not well understood. We investigated the endogenous effect of these CREs with CRISPR (clustered regularly interspaced short palindromic repeats) disruptions and observed GJB2 expression. To decipher the GJB2 regulatory landscape, we used the 4C-seq technique and defined new chromatin contacts inside the DFNB1 locus, which permit DNA loops and long-range regulation. Moreover, through ChIP-PCR, we determined the involvement of the MEIS1 transcription factor in GJB2 expression. Taken together, the results of our study enable us to describe the 3D DFNB1 regulatory landscape.

Meis1 plays roles in cortical development through regulation of cellular proliferative capacity in the embryonic cerebrum.

Meis1 (myeloid ecotropic insertion site 1) is known to be related to embryonic development and cancer. In this study, to analyze the function of Meis1 in neural stem cells, we crossed Meis1fl/fl (Meis1 floxed) mice with Nestin-Cre mice. The results showed that Meis1-conditional knockout mice showed cerebral cortex malformation. The mice had a significantly thinner cortex than wildtype mice. At E14.5, BrdU incorporation and Pax6-positive radial glial cells were significantly decreased in the cerebral cortex of Meis1 knockout embryos as compared with wild-type embryos, whereas Tbr2-positive intermediate progenitors and NeuN-positive differentiated neurons were not. Cell death detected by immunostaining with cleaved caspase3 antibody showed no difference in the cortex between knockout and wild-type embryos. Furthermore, knockout of Meis1 in embryo by in utero electroporation showed that cellular migration was disturbed during cortical development. Therefore, Meis1 could play important roles during cortical development through the regulation of cell proliferation and migration in the embryonic cerebral cortex.

Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc.

The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 alone immortalizes cells, cooperation with Meis1 is necessary to induce a full leukemic phenotype. Here, we applied degron techniques to elucidate the leukemogenic contribution of Meis1. Chromatin immunoprecipitation experiments revealed that Meis1 localized mainly to H3K27 acetylated and H3K4 mono-methylated enhancers preactivated by HoxA9. Chromatin association of Meis1 required physical presence of HoxA9 and all Meis1 DNA interactions were rapidly lost after HoxA9 degradation. Meis1 controlled a gene expression pattern dominated by Myc, ribosome biogenesis and ribosomal RNA synthesis genes. While Myc accounted for the cell cycle stimulating effect of Meis1, overexpression of this oncogene alone did not accelerate leukemogenesis. Besides its effect on Myc, Meis1 induced transcription of ribosomal biogenesis genes. This was accompanied by an elevated resistance against inhibition of ribosomal RNA synthesis and translation, but without affecting steady-state protein synthesis. Finally, we demonstrate that HoxA9 and Meis1 proteins are stabilized by post-translational modification. Mutation of HoxA9/Meis1 phosphorylation sites or inhibition of casein kinase 2 lead to rapid protein degradation suggesting a potential pathway for pharmacological intervention.