Zinc Finger DNA-binding Protein Research Articles

Zinc finger protein 521 (ZNF521/Zfp521): Insights into its structure, function, regulation, and significance to cell differentiation

The zinc finger protein 521 (ZNF521) gene is located on human chromosome 18, specifically in the 18q11.2 region. As a zinc finger DNA-binding protein, ZNF521/Zfp521 acts as a transcription cofactor involved in the regulation of cell differentiation across various cell types, including hematopoietic, neural, and mesenchyme stem cells, B progenitor cells, preadipocytes, and osteoblasts. Its interactions with key proteins such as RUNX2, EBF1, SIAH2, or BMP2 further modulate these processes. In this review, we briefly summarize the current understanding of ZNF521/Zfp521, encompassing its structural, functional, post-translational modification, and cellular signaling pathways, especially its roles in cell differentiation and related diseases. In addition, we explore the process and effects of the interaction between Zfp521 and other proteins and discuss the molecular mechanisms of its roles in adipogenic, osteogenic, chondrogenic, and neural differentiation.

Multifaceted roles of IKZF1 gene, perspectives from bench to bedside.

The IKZF1 gene encodes a transcription factor that belongs to the family of zinc-finger DNA-binding proteins associated with chromatin remodeling. The protein product, IKAROS, had been proved to regulate lymphopoiesis. Subsequent mouse model studies have further confirmed its regulating role in lymphopoiesis as well as in hematopoiesis; besides, it associates with immune function, certain immune disorders like common variable immunodeficiency and dysgammaglobulinemia have been proved to be associated with germline IKZF1 mutations. Dysfunction of IKAROS also bears paramount significance in leukemic transformation and alterations of IKZF1 gene predicts a poor prognosis in hematological malignancies. As an independent prognostic marker, IKZF1 has been incorporated in the risk stratification of BCP-ALL and stratification-guided therapy has also been generated. In this review, we provide a concise and comprehensive overview on the multifaceted roles of IKZF1 gene.

IKZF1 Alterations and Therapeutic Targeting in B-Cell Acute Lymphoblastic Leukemia.

IKZF1 encodes the transcription factor IKAROS, a zinc finger DNA-binding protein with a key role in lymphoid lineage development. IKAROS plays a critical role in the development of lineage-restricted mature lymphocytes. Deletions within IKZF1 in B-cell acute lymphoblastic leukemia (B-ALL) lead to a loss of normal IKAROS function, conferring leukemic stem cell properties, including self-renewal and subsequent uncontrolled growth. IKZF1 deletions are associated with treatment resistance and inferior outcomes. Early identification of IKZF1 deletions in B-ALL may inform the intensification of therapy and other potential treatment strategies to improve outcomes in this high-risk leukemia.

Dynamics and genetic regulation of macronutrient concentrations during grain development in maize

Nitrogen (N), phosphorus (P), and potassium (K) are essential macronutrients that are crucial not only for maize growth and development, but also for crop yield and quality. The genetic basis of macronutrient dynamics and accumulation during grain filling in maize remains largely unknown. In this study, we evaluated grain N, P, and K concentrations in 206 recombinant inbred lines generated from a cross of DH1M and T877 at six time points after pollination. We then calculated conditional phenotypic values at different time intervals to explore the dynamic characteristics of the N, P, and K concentrations. Abundant phenotypic variations were observed in the concentrations and net changes of these nutrients. Unconditional quantitative trait locus (QTL) mapping revealed 41 non-redundant QTLs, including 17, 16, and 14 for the N, P, and K concentrations, respectively. Conditional QTL mapping uncovered 39 non-redundant QTLs related to net changes in the N, P, and K concentrations. By combining QTL, gene expression, co-expression analysis, and comparative genomic data, we identified 44, 36, and 44 candidate genes for the N, P, and K concentrations, respectively, including GRMZM2G371058 encoding a Dof-type zinc finger DNA-binding family protein, which was associated with the N concentration, and GRMZM2G113967 encoding a CBL-interacting protein kinase, which was related to the K concentration. The results deepen our understanding of the genetic factors controlling N, P, and K accumulation during maize grain development and provide valuable genes for the genetic improvement of nutrient concentrations in maize.

Comparative Analysis of the GATA Transcription Factors in Five Solanaceae Species and Their Responses to Salt Stress in Wolfberry (Lycium barbarum L.).

GATA proteins are a class of zinc-finger DNA-binding proteins that participate in diverse regulatory processes in plants, including the development processes and responses to environmental stresses. However, a comprehensive analysis of the GATA gene family has not been performed in a wolfberry (Lycium barbarum L.) or other Solanaceae species. There are 156 GATA genes identified in five Solanaceae species (Lycium barbarum L., Solanum lycopersicum L., Capsicum annuum L., Solanum tuberosum L., and Solanum melongena L.) in this study. Based on their phylogeny, they can be categorized into four subfamilies (I-IV). Noticeably, synteny analysis revealed that dispersed- and whole-genome duplication contributed to the expansion of the GATA gene family. Purifying selection was a major force driving the evolution of GATA genes. Moreover, the predicted cis-elements revealed the potential roles of wolfberry GATA genes in phytohormone, development, and stress responses. Furthermore, the RNA-seq analysis identified 31 LbaGATA genes with different transcript profiling under salt stress. Nine candidate genes were then selected for further verification using quantitative real-time PCR. The results revealed that four candidate LbaGATA genes (LbaGATA8, LbaGATA19, LbaGATA20, and LbaGATA24) are potentially involved in salt-stress responses. In conclusion, this study contributes significantly to our understanding of the evolution and function of GATA genes among the Solanaceae species, including wolfberry.

Genome-wide identification, evolution and expression pattern analysis of the GATA gene family in Sorghum bicolor.

The GATA family of transcription factors is zinc finger DNA binding proteins involved in a variety of biological processes, including plant growth and development and response to biotic/abiotic stresses, and thus play an essential role in plant response to environmental changes. However, the GATA gene family of Sorghum (SbGATA) has not been systematically analyzed and reported yet. Herein, we used a variety of bioinformatics methods and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to explore the evolution and function of the 33 SbGATA genes identified. These SbGATA genes, distributed on 10 chromosomes, are classified into four subfamilies (I-IV) containing one pair of tandem duplications and nine pairs of segment duplications, which are more closely related to the monocot Brachypodium distachyon and Oryza sativa GATA genes. The physicochemical properties of the SbGATAs are significantly different among the subfamilies, while the protein structure and conserved protein motifs are highly conserved in the subfamilies. In addition, the transcription of SbGATAs is tissue-specific during Sorghum growth and development, which allows for functional diversity in response to stress and hormones. Collectively, our study lays a theoretical foundation for an in-depth analysis of the functions, mechanisms and evolutionary relationships of SbGATA during plant growth and development.

Functional prediction of tomato PLATZ family members and functional verification of SlPLATZ17

PLATZ is a novel zinc finger DNA-binding protein that plays an important role in regulating plant growth and development and resisting abiotic stress. However, there has been very little research on the function of this family gene in tomatoes, which limits its application in germplasm resource improvement. Therefore, the PLATZ gene family was identified and analyzed in tomato, and its roles were predicted and verified to provide a basis for in-depth research on SlPLATZ gene function. In this study, the PLATZ family members of tomato were identified in the whole genome, and 19 SlPLATZ genes were obtained. Functional prediction was conducted based on gene and promoter structure analysis and RNA-seq-based expression pattern analysis. SlPLATZ genes that responded significantly under different abiotic stresses or were significantly differentially expressed among multiple tissues were screened as functional gene resources. SlPLATZ17 was selected for functional verification by experiment-based analysis. The results showed that the downregulation of SlPLATZ17 gene expression reduced the drought and salt tolerance of tomato plants. Tomato plants overexpressing SlPLATZ17 had larger flower sizes and long, thin petals, adjacent petals were not connected at the base, and the stamen circumference was smaller. This study contributes to understanding the functions of the SlPLATZ family in tomato and provides a reference for functional gene screening.

Dimerization choice and alternative functions of ZBTB transcription factors.

Zinc Finger DNA-binding domain-containing proteins are the most populous family among eukaryotic transcription factors. Among these, members of the BTB domain-containing ZBTB sub-family are mostly known for their transcriptional repressive functions. In this Viewpoint article, we explore molecular mechanisms that potentially diversify the function of ZBTB proteins based on their homo and heterodimerization, alternative splicing and post-translational modifications. We describe how the BTB domain is as much a scaffold for the assembly of co-repressors, as a domain that regulates protein stability. We highlight another mechanism that regulates ZBTB protein stability: phosphorylation in the zinc finger domain. We explore the non-transcriptional, structural roles of ZBTB proteins and highlight novel findings that describe the ability of ZBTB proteins to associate with poly adenosine ribose in the nucleus during the DNA damage response. Herein, we discuss the contribution of BTB domain scaffolds to the formation of transcriptional repressive complexes, to chromosome compartmentalization and their non-transcriptional, purely structural functions in the nucleus.

KLF6 alleviates hepatic ischemia-reperfusion injury by inhibiting autophagy

Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs) constitute a family of C2/H2 zinc finger DNA-binding proteins. KLF6, a member of the KLF protein family, plays crucial roles in proliferation, metabolism, inflammation, and injury responses; however, its role in HIR is largely remains unknown. After I/R injury, we found that KLF6 expression in mice and hepatocytes was significantly upregulated. Mice were then subjected to I/R following injection of shKLF6- and KLF6-overexpressing adenovirus through the tail vein. KLF6 deficiency markedly exacerbated liver damage, cell apoptosis, and activation of hepatic inflammatory responses, whereas hepatic overexpression of KLF6 in mice produced the opposite results. In addition, we knocked out or overexpressed KLF6 in AML12 cells before exposing them to a hypoxia-reoxygenation challenge. KLF6 knockout decreased cell viability and increased hepatocyte inflammation, apoptosis, and ROS, whereas KLF6 overexpression had the opposite effects. Mechanistically, KLF6 inhibited the overactivation of autophagy at the initial stage, and the regulatory effect of KLF6 on I/R injury was autophagy-dependent. CHIP-qPCR and luciferase reporter gene assays confirmed that KLF6 bound to the promoter region of Beclin1 and inhibited its transcription. Additionally, KLF6 activated the mTOR/ULK1 pathway. Finally, we performed a retrospective analysis of the clinical data of liver transplantation patients and identified significant associations between KLF6 expression and liver function following liver transplantation. In conclusion, KLF6 inhibited the overactivation of autophagy via transcriptional regulation of Beclin1 and activation of the mTOR/ULK1 pathway, thereby protecting the liver from I/R injury. KLF6 is expected to serve as a biomarker for estimating the severity of I/R injury following liver transplantation.

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.

Deleterious Rare Mutations of GLI1 Dysregulate Sonic Hedgehog Signaling in Human Congenital Heart Disease.

The Glioma-associated oncogene (Gli) family members of zinc finger DNA-binding proteins are core effectors of Sonic hedgehog (SHH) signaling pathway. Studies in model organisms have identified that the Gli genes play critical roles during organ development, including the heart, brain, kidneys, etc. Deleterious mutations in GLI genes have previously been revealed in several human developmental disorders, but few in congenital heart disease (CHD). In this study, the mutations in GLI1-3 genes were captured by next generation sequencing in human cohorts composed of 412 individuals with CHD and 213 ethnically matched normal controls. A total of 20 patient-specific nonsynonymous rare mutations in coding regions of human GLI1-3 genes were identified. Functional analyses showed that GLI1 c.820G> T (p.G274C) is a gain-of-function mutation, while GLI1 c.878G>A (p.R293H) and c.1442T>A (p.L481X) are loss-of-function mutations. Our findings suggested that deleterious rare mutations in GLI1 gene broke the balance of the SHH signaling pathway regulation and may constitute a great contribution to human CHD, which shed new light on understanding genetic mechanism of embryo cardiogenesis regulated by SHH signaling.

Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddAtox, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Because ZFDs, unlike CRISPR-derived base editors, do not cleave DNA to yield single- or double-strand breaks, no unwanted indels caused by error-prone non-homologous end joining are produced at target sites. Furthermore, recombinant ZFD proteins, expressed in and purified from E. coli, penetrate cultured human cells spontaneously to induce targeted base conversions, demonstrating the proof-of-principle of gene-free gene therapy.

HIC2 Controls Developmental Hemoglobin Switching By Repressing BCL11A Transcription

One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin production in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease (SCD) and thalassemia. BCL11A is a transcriptional repressor that is thought to directly silence the fetal β-type globin (HBG1/2) genes in adult erythroid cells. Transcriptome and RNA polymerase II profiling indicate that the BCL11A gene is transcribed considerably more highly in adult erythroblasts compared to fetal cells, accounting in large part for corresponding changes in BCL11A protein levels. Yet, the mechanism governing BCL11A developmental regulation is still unclear.To identify novel regulators of the fetal-to-adult globin switch, we interrogated our recent CRISPR based genetic screens that employed single guide RNAs (sgRNAs) targeting transcription factors (Huang et al., Blood, 2020) and uncovered HIC2, a penta-dactyl zinc finger DNA binding protein bearing a BTB/POZ domain as a novel regulator of hemoglobin switching.HIC2 is expressed more highly in fetal erythroblasts compared to adult cells, a pattern inverse to that of BCL11A. Overexpression (OE) of HIC2 in the adult type erythroid HUDEP2 cell line stimulated the expression of 322 genes while impairing that of 224 genes (FDR < 0.01 and fold change ≥ 2). The most highly upregulated genes (>150-fold) were HBG1/2. Upregulation was accompanied by gains in chromatin accessibility and histone H3K27acetylation of HBG1/2, and increased chromatin contacts between the distal globin gene enhancer (LCR) and the HBG1/2 genes. Overexpression of HIC2 in primary human erythroblasts also significantly increased HBG1/2 mRNA and protein levels, sufficient to reduce cell sickling in SCD patient-derived erythroid cells.HIC2 OE lowered BCL11A mature and pre-mRNA production, indicating that HIC2 attenuates BCL11A transcription. Forced expression of BCL11A restored HBG1/2 silencing in HIC2 OE HUDEP2 cells, suggesting that BCL11A repression accounts for the effects of HIC2 on fetal globin genes. ChIP-seq revealed a strong HIC2 binding peak at the erythroid BCL11A +55 enhancer. HIC2 OE reduced chromatin accessibility and H3K27acetylation of the +55 enhancer, as well as the enhancer-promoter contacts, suggesting that HIC2 directly decommissions the enhancer to attenuate BCL11A transcription.The BCL11A +55 enhancer contains two consensus HIC2 binding motifs under the HIC2 peak adjacent to GATA:E-box and GATA motifs. CRISPR-mediated mutagenesis of both HIC2 motifs raised BCL11A basal level transcription and diminished the ability of overexpressed HIC2 to repress BCL11A transcription. Notably, HIC2 OE impaired binding of transcription factor GATA1 at the +55 enhancer, suggesting that this enhancer is under developmental control. Indeed, GATA1 binding and chromatin accessibility of +55 enhancer were virtually undetectable in HUDEP1 cells, which represent a more fetal-like state. CRISPR-mediated depletion of HIC2 in HUDEP1 cells reversed this pattern with gains in GATA1 binding, chromatin accessibility, and BCL11A transcription.In sum, HIC2 emerges as a critical regulator of hemoglobin switching that operates by imposing developmental stage-specific control onto a BCL11A transcriptional enhancer. DisclosuresBlobel: Fulcrum therapeutics: Consultancy; Pfizer: Research Funding.

Graphene Oxide-Based Simple and Rapid Detection of Antibiotic Resistance Gene via Quantum Dot-Labeled Zinc Finger Proteins.

With the increasing rise of antibiotic-resistant pathogens, a simple and rapid detection of antibiotic resistance gene (ARG) is crucial to mitigate the spreading of antibiotic resistance. DNA-binding zinc finger proteins (ZFPs) can be engineered to recognize specific double-stranded (ds) DNA sequences in ARG. Here, we designed a simple and rapid method to detect ARG in bacteria utilizing engineered ZFPs and 2D nanosheet graphene oxide (GO) as a sensing platform. Our approach relies on the on and off effect of fluorescence signal in the presence and absence of target ARG, respectively. By taking advantage of the unique quenching capability of GO due to its electronic property, quantum dot (QD)-labeled ZFPs are adsorbed onto the GO sheets, and their fluorescence signal is quenched by proximal GO sheets through fluorescence resonance energy transfer (FRET). In the presence of target DNA, ZFP binding to the target DNA induces dissociation from GO, thereby restoring the fluorescence signal. Our system detects target DNA through restoration of QD emission as the restored signal increases directly with target DNA concentrations. Engineered ZFPs were able to detect specific dsDNA of the tetracycline resistance gene tetM with high specificity after only 10 min incubation on our GO-based sensing system. Our sensing system employed one-step FRET-based ZFP and GO combined technology to enable rapid and quantitative detection of ARG, providing a limit of detection as low as 1 nM. This study demonstrated the application of GO in conjunction with engineered DNA-binding domains for the direct detection of dsDNA with great potential as a rapid and reliable screening and detecton method against the growing threat of antibiotic resistant bacteria.

IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK.

Interferon regulatory factor 4 (IRF4) is a transcriptional regulator of immune system development and function. Here, we investigated the role of IRF4 in controlling responsiveness to B-cell receptor (BCR) stimulation in chronic lymphocytic leukemia (CLL). We modulated IRF4 levels by transfecting CLL cells with an IRF4 vector or by silencing using small-interfering RNAs. Higher IRF4 levels attenuated BCR signaling by reducing AKT and ERK phosphorylation and calcium release. Conversely, IRF4 reduction improved the strength of the intracellular cascade activated by BCR engagement. Our results also indicated that IRF4 negatively regulates the expression of the spleen tyrosine kinase SYK, a crucial protein for propagation of BCR signaling, and the zinc finger DNA-binding protein IKAROS. We modulated IKAROS protein levels both by genetic manipulation and pharmacologically by treating CLL cells with lenalidomide and avadomide (IMIDs). IKAROS promoted BCR signaling by reducing the expression of inositol 5-phosphatase SHIP1. Lastly, IMIDs induced IRF4 expression, while down-regulating IKAROS and interfered with survival advantage mediated by BCR triggering, also in combination with ibrutinib. Overall, our findings elucidate the mechanism by which IRF4 tunes BCR signaling in CLL cells. Low IRF4 levels allow an efficient transmission of BCR signal throughout the accumulation of SYK and IKAROS.

KRAB domain of ZFP568 disrupts TRIM28-mediated abnormal interactions in cancer cells.

Interactions of KRAB (Krüppel-associated box)-associated protein KAP1 [also known as TRIM28 (tripartite motif containing protein 28)] with DNA-binding KRAB zinc finger (KRAB-ZF) proteins silence many transposable elements during embryogenesis. However, in some cancers, TRIM28 is upregulated and interacts with different partners, many of which are transcription regulators such as EZH2 in MCF7 cells, to form abnormal repressive or activating complexes that lead to misregulation of genes. We ask whether a KRAB domain—the TRIM28 interaction domain present in native binding partners of TRIM28 that mediate repression of transposable elements—could be used as a tool molecule to disrupt aberrant TRIM28 complexes. Expression of KRAB domain containing fragments from a KRAB-ZF protein (ZFP568) in MCF7 cells, without the DNA-binding zinc fingers, inhibited TRIM28–EZH2 interactions and caused degradation of both TRIM28 and EZH2 proteins as well as other components of the EZH2-associated polycomb repressor 2 complex. In consequence, the product of EZH2 enzymatic activity, trimethylation of histone H3 lysine 27 level, was significantly reduced. The expression of a synthetic KRAB domain significantly inhibits the growth of breast cancer cells (MCF7) but has no effect on normal (immortalized) human mammary epithelial cells (MCF10a). Further, we found that TRIM28 is a positive regulator of TRIM24 protein levels, as observed previously in prostate cancer cells, and expression of the KRAB domain also lowered TRIM24 protein. Importantly, reduction of TRIM24 levels, by treatment with either the KRAB domain or a small-molecule degrader targeted to TRIM24, is accompanied by an elevated level of tumor suppressor p53. Taken together, this study reveals a novel mechanism for a TRIM28-associated protein stability network and establishes TRIM28 as a potential therapeutic target in cancers where TRIM28 is elevated. Finally, we discuss a potential mechanism of KRAB-ZF gene expression controlled by a regulatory feedback loop of TRIM28–KRAB.

Pioneering meiotic recombination.

To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.

GATA2 mutations in myeloid malignancies: Two zinc fingers in many pies

The GATA family of transcription factors are zinc finger (ZF) DNA-binding proteins that regulate transcription during development and cell differentiation. GATA2 plays an essential role in the regulation of hematopoiesis. As a result, mutations in this gene or alterations in its expression level or function have been linked to a variety of human hematologic disorders. In this review, we summarize the findings and developments over the recent years regarding the clinical correlations and functional properties of distinct GATA2 mutations in hematopoietic malignancies, with particular focus on the mutational hotspots in the ZF domains.

CK2 Inhibitor CX4945 Shows Strong In Vivo Synergistic Anti-Leukemia Activity with Rapamycin in B-ALL Due to Enhanced Ikaros Mediated Repression of mTOR Pathway

Casein Kinase II (CK2) is a pro-oncogenic protein that is overexpressed in various cancers including leukemia. Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros activity (genetic alteration and functional inactivation) in high-risk B-ALL is associated with resistance to conventional chemotherapy and a poor prognosis. Ikaros binds to the promoters and/or upstream regulatory sequences of its target genes and regulates their transcription via chromatin remodeling. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions and interferes with Ikaros' ability to repress cell cycle progression and the PI3K pathway. Targeted inhibition of CK2 using specific inhibitor CX4945 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. CX4945 showed strong in vivo anti-leukemia effect in high risk B-ALL patient derived xenograft models (Song et al, Blood 2015). CX4945 is currently in clinical trial for solid tumors. mTOR is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Rapamycin is a specific inhibitor of mTOR that is currently in clinical trial for the treatment of relapsed/refractory leukemia. Here we report in vivo efficacy of combination therapy of rapamycin with CX4945 using high risk B-ALL pre-clinical models and describe mechanism of synergistic action of CX4945 and rapamycin. Methods and results: The therapeutic effect of the CX-4945, in combination with rapamycin was tested using three different patient-derived xenografts (PDX) from patients with high-risk B-ALL and its efficacy will be compared to single-drug treatment. All these leukemia samples were obtained from high risk pediatric B-ALL patients with high CK2 expression and one had IKZF1 haplo - deficiency. After engraftment following tail vein inoculation of leukemia cells into immunocompromised (NRG) mice, mice were treated with vehicle, rapamycin or CX4945 alone or combination of both CX4945 and rapamycin. Anti-tumor effect was measured by quantifying leukemia burden in bone marrow, spleen and peripheral blood by cell count and immune-phenotyping using flow cytometry. Overall survival was measured using Kaplan -Myers analysis in another cohort after similar treatment regimen. The analysis of Ikaros' global genome-wide occupancy in the human B-ALL cell line, Nalm6, and in primary cells from patient with B-ALL, showed that Ikaros binds to the upstream regulatory element (URE) of MTOR gene. Further, Ikaros binding to MTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating mTOR transcription in Nalm-6 B-ALL cell line was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA results in increased transcription of mTOR whereas overexpression of Ikaros by transduction of Nalm-6 cells with Ikaros-containing retrovirus resulted in sharp reduction of mTOR expression. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR. Over-expression of CK2 via retroviral transduction resulted in increased transcription of the mTOR mRNA, as measured by qRT-PCR, as well as increased overall expression of mTOR protein, as measured by western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the MTOR gene promoter. CK2 inhibition was also associated with reduced histone H3K9ac and H3K4me3 marks at the mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In summary, these results establish in vivo anti-leukemia effect of combination therapy using rapamycin and CX4945 in high risk B-ALL PDX models. CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for evaluating clinical use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. The results of our study establish a new paradigm for targeting the transcriptional network that regulates expression of the gene in hyperactive oncogenic signaling pathways such as mTOR. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Role of GFI1 in Epigenetic Regulation of MDS and AML Pathogenesis: Mechanisms and Therapeutic Implications.

Growth factor independence 1 (GFI1) is a DNA binding zinc finger protein, which can mediate transcriptional repression mainly by recruiting histone-modifying enzymes to its target genes. GFI1 plays important roles in hematopoiesis, in particular by regulating both the function of hematopoietic stem- and precursor cells and differentiation along myeloid and lymphoid lineages. In recent years, a number of publications have provided evidence that GFI1 is involved in the pathogenesis of acute myeloid leukemia (AML), its proposed precursor, myelodysplastic syndrome (MDS), and possibly also in the progression from MDS to AML. For instance, expression levels of the GFI1 gene correlate with patient survival and treatment response in both AML and MDS and can influence disease progression and maintenance in experimental animal models. Also, a non-synonymous single nucleotide polymorphism (SNP) of GFI1, GFI1-36N, which encodes a variant GFI1 protein with a decreased efficiency to act as a transcriptional repressor, was found to be a prognostic factor for the development of AML and MDS. Both the GFI1-36N variant as well as reduced expression of the GFI1 gene lead to genome-wide epigenetic changes at sites where GFI1 occupies target gene promoters and enhancers. These epigenetic changes alter the response of leukemic cells to epigenetic drugs such as HDAC- or HAT inhibitors, indicating that GFI1 expression levels and genetic variants of GFI1 are of clinical relevance. Based on these and other findings, specific therapeutic approaches have been proposed to treat AML by targeting some of the epigenetic changes that occur as a consequence of GFI1 expression. Here, we will review the well-known role of Gfi1 as a transcription factor and describe the more recently discovered functions of GFI1 that are independent of DNA binding and how these might affect disease progression and the choice of epigenetic drugs for therapeutic regimens of AML and MDS.