Kruppel-associated Box Domain Research Articles

ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178–449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.

Promoter hypermethylation and comprehensive regulation of ncRNA lead to the down-regulation of ZNF880, providing a new insight for the therapeutics and research of colorectal cancer

The human genome encodes more than 350 kinds of Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), KRAB-type ZNF transcription factor family (KZNF) plays a vital role in gene regulatory networks. The KZNF family members include a large number of highly homologous genes, gene subtypes and pseudogenes, and their expression has a high degree of tissue specificity and precision. Due to the high complexity of its regulatory network, the KZNF gene family has not been researched in sufficient, and the role of its members in the occurrence of cancer is mostly unexplored. In this study, ZNF880 was significantly associated with overall survival (OS) and disease-free survival (DFS) in colorectal carcinoma (CRC) patients. Low ZNF880 expression resulted in shorter OS and DFS. Combined with Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) data collection in the TCGA database, we found that ZNF880 was significantly down-regulated in CRC. Further analysis of the sequence variation of ZNF880 in CRC showed that ZNF880 accumulated a large number of SNV in the C2H2 domain and KRAB domain, while promoter region of ZNF880 also showed high methylation in COAD and READ. Combined with the Cbioportal and TIMER databases, the expression of mutant ZNF880 was significantly lower in COAD compared to the wild type. Simultaneously, the lncRNA-miRNA-ZNF880 ceRNA regulatory network was constructed through co-expression and miRNAs target gene prediction, demonstrating the precision of the ZNF880 regulatory network. In addition, the decreased expression of ZNF880 caused the significant immune infiltration decreases of CD8 + cells in COAD. In contrast, the immune infiltration of CD4 + cells and macrophages in COAD is positively correlated with ZNF880. Finally, through protein–protein interaction (PPI) network analysis and transcription factor target gene prediction, we screened out the genes most likely to be related to the function of ZNF880. CENPK, IFNGR2, REC8 and ZBTB17 were identified as the most closely functioning genes with ZNF880, which may indicate that ZNF880 has important links with the formation of cell centromere, tumor immunity, cell cycle and other pathways closely related to the occurrence of CRC. These studies show that the down-regulation of ZNF880 gene is closely related to CRC, and the targeted change of the expression of its regulatory molecules (miRNA and lncRNA) may be a new perspective for CRC treatment.

High Expression of POGK Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma.

Kruppel-associated box (KRAB) proteins reportedly play a dual role in neoplastic transformation. At present, little is known about the function of the proteins encoded by the human pogo transposable element derived with KRAB domain (POGK) gene. Herein, we evaluated the prognostic significance of POGK expression in patients with hepatocellular carcinoma (HCC). The data of HCC patients was downloaded from The Cancer Genome Atlas (TCGA) database. To determine the relationship between POGK and clinical features, logistic regression was applied. Cox regression and Kaplan-Meier analyses were used to evaluate the correlation between POGK and survival rates. Gene ontology (GO) analysis and Gene set enrichment analysis (GSEA) were conducted to identify the enriched pathways and functions associated with POGK. A total of 374 HCC patients were identified in TCGA. POGK was significantly upregulated in HCC and correlated with tumor status (p = 0.036), race (p = 0.025), weight (p = 0.002), body mass index (p = 0.033), histologic grade (p < 0.001), and alpha-fetoprotein (p < 0.001). High POGK expression in HCC patients correlated with a poor outcome in terms of overall survival (p = 0.0018), progression-free survival (p = 0.0087), relapse-free survival (p = 0.045), and disease-specific survival (p = 0.014), according to Kaplan-Meier analysis. Receiver operating characteristic curve analysis showed that the area under the curve of POGK expression for HCC diagnosis was 0.891. GSEA showed that high POGK expression might activate mitotic prometaphase, kinesins, homologous DNA pairing and strand exchange, MET activates PTK2 signaling pathway, G1 to S cell cycle control, Aurora B pathway, ncRNAs involved in WNT signaling pathway, hepatitis C, and ncRNAs involved in the STAT3 signaling pathway. POGK expression correlated with the abundance of adaptive and innate immunocytes in HCC. High expression of POGK has high diagnostic and prognostic values in patients with HCC. Moreover, POGK expression is correlated with immune infiltration in HCC.

Poly zinc finger protein ZFP14 suppresses lymphomagenesis and abnormal inflammatory response via the HOXA gene cluster

Poly zinc finger proteins (ZFP) that contain a KRAB (Krüppel-associated box) domain represent the largest class of transcription factors in higher organisms, but their roles in development and pathogenesis are largely undefined. ZFP14 (also known as ZNF531) contains thirteen zinc fingers and is highly conserved across species. Notably, we found that ZFP14 is frequently down-regulated in a multitude of human cancers, which correlates with poor prognosis of patients. Since ZFP14 has never been characterized, we generated a Zfp14-deficient mouse model to investigate the role of ZFP14 in development and pathogenesis. We showed that the mice deficient in Zfp14 had a short lifespan and were prone to diffuse large B-cell lymphoma (DLBCL), hyperplasia in multiple organs, systemic chronic inflammation, liver steatosis, and pancreatitis. Additionally, several pro-inflammatory cytokines, including IL-1β, IL18, and TNFα, were highly expressed in inflamed Zfp14−/− mice spleens, livers, kidneys and lungs. To determine the underlying mechanism, RNA-seq analysis was performed and showed that the loss of ZFP14 led to increased expression of inflammatory and tumor-promoting genes. Out of the various tumor-promoting genes upregulated by ZFP14 loss, the HOXA gene cluster, which is known to promote lymphomagenesis and conserved between mouse and human, is consistently induced by loss of ZFP14. Moreover, we showed that the HOXA gene expression was inversely correlated with that of ZFP14 in human cancer patients and higher HOXA1 expression was correlated with poor patient prognosis. Together, we postulate that ZFP14 suppresses lymphomagenesis and abnormal inflammatory response by maintaining proper expression of the HOXA gene cluster.

DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.

The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A-/-, Dnmt3b-/-) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings.

The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain.

Krüppel-associated box (KRAB) zinc finger proteins are a large class of tetrapod transcription factors that usually exert transcriptional repression through recruitment of TRIM28/KAP1. The evolutionary root of modern KRAB domains (mKRAB) can be traced back to an ancestral motif (aKRAB) that occurs even in invertebrates. Here, we first stratified three subgroups of aKRAB sequences from the animal kingdom (PRDM9, SSX and coelacanth KZNF families) and defined ancestral subdomains for KRAB-A and KRAB-B. Using human ZNF10 mKRAB-AB as blueprints for function, we then identified the necessary amino acid changes that transform the inactive aKRAB-A of human PRDM9 into an mKRAB domain capable of mediating silencing and complexing TRIM28/KAP1 in human cells when employed as a hybrid with ZNF10-B. Full gain of function required replacement of residues KR by the conserved motif MLE (positionsA32-A34), which inserted an additional residue, and exchange of A9/S for F, A20/M for L, and A27/R for V. AlphaFold2 modelling documented an evolutionary conserved L-shaped body of two α-helices in all KRAB domains. It is transformed into a characteristic spatial arrangement typical for mKRAB-AB upon the amino acid replacements and in conjunction with a third helix supplied by mKRAB-B. Side-chains pointing outward from the core KRAB 3D structure may reveal a protein-protein interaction code enabling graded binding of TRIM28 to different KRAB domains. Our data provide basic insights into structure-function relationships and emulate transitions of KRAB during evolution.

An efficient KRAB domain for CRISPRi applications in human cells.

Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently than existing platforms.

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.

19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer.

Zinc-finger proteins (ZFPs) are the largest transcription factor family in mammals, involved in the regulation of multiple physiologic processes including cell differentiation, proliferation, apoptosis and neoplastic transformation. Approximately one-third of ZFPs are Krüppel-associated box domain (KRAB)-ZFPs.Methods: ZNF471 expression and methylation were detected by reverse-transcription PCR and methylation-specific PCR. The impact and mechanism of ectopic ZNF471 expression in esophageal squamous cell carcinoma (ESCC) cells was evaluated in vitro and in vivo.Results: We identified a 19q13 KRAB-ZFP, ZNF471, as a methylated target in ESCC. We further found that ZNF471 is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues, due to its aberrant promoter CpG methylation, and further confirmed by methylation analysis and treatment with demethylation agent. Restoration of ZNF471 expression in silenced ESCC cells significantly altered cell morphology, induced apoptosis and G0/G1 arrest, and inhibited tumor cell colony formation, viability, migration and invasion. Importantly, ZNF471 was found to activate the expression of MAPK10/JNK3 and PCDH family genes, and further enhance MAPK10 signaling and downstream gene expression through binding to the MAPK10/JNK3 promoter.Conclusion: Our results demonstrate that ZNF471 is an important tumor suppressor and loss of ZNF471 functions hampers MAPK10/JNK3 signaling during esophageal carcinogenesis.

DUF3669, a \u201cdomain of unknown function\u201d within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression

BackgroundZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the “domain of unknown function 3669” (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson’s disease.ResultsHere we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669’s impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved “MLE” sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein.ConclusionDUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

Colla corii asini might upregulate ZNF471 and THOC5 by KRAB domain-containing zinc-finger protein pathway and THO complex subunit 5 pathway to improve anemia of pregnant women with β-thalassemia.

Pregnant patients with β-thalassemia are more likely to have progressive anemia which expose them to risk of adverse pregnancy outcomes, blood transfusion, and iron overload. Results from our previous study indicated that Colla corii asini (CCA, E'jiao), a natural ingredient of traditional Chinese medicine, could significantly increase hemoglobin level of pregnant women with β- thalassemia, but the underlying molecular mechanism was unclear. Thus, we applied high-throughput transcriptome sequencing to study the transcriptomic change before and after the CCA treatment. Twenty eligible pregnant women were recruited and randomized to either the CCA treatment group or the blank control group in a 3:1 ratio. Patients in the treatment group orally received daily 15g CCA powder for 4weeks. We analyzed the therapeutic effect indexes and the transcriptomic change in subjects' peripheral blood before and after treatment. We found that β CD 41-42(-TTCT)/βA was the main genotype of the subjects. The regulatory impact of CCA treatment became more evident among the subjects of genotype β CD 41-42(-TTCT)/βA. Gene ontogenesis analysis revealed that the top five molecular functions of differentially expressed genes were involved in membrane functionality and cellular structure. We further identified two consistent upregulated genes ZNF471 and THOC5 in the effective treatment group, which were engaged in Kruppel-associated box (KRAB) domain-containing zinc-finger protein pathway and THOC5 pathway, respectively. Based on our current findings, we hypothesize that the anti-anemia effect of CCA on pregnant women with β-thalassemia might be related to translation regulation of spectrin synthesis, membrane stability, and eventually prolonged the life span of erythrocytes.

A4617 GENETIC VARIATIONS AMONG PATIENTS WITH DILATED CARDIOMYOPATHY IN MALAYSIA

Objectives: To identify the genetic variations of dilated cardiomyopathy (DCM) in Malaysia population Methods: Two thousand and eleven subjects (1,978 males and 33 females) were recruited and screened by echocardiography from various community health screening programs (n = 1982) and hospital-based patients (n = 29). Thirty patients (27 males and 3 females) diagnosed with DCM were recruited of which, one subject was recruited from community health screening program and 29 were recruited from hospital-based admission. Written informed consent was obtained prior to commencement of this study. Echocardiogram (ECHO) performed on the recruited subjects. Diagnosis of DCM is based on a left ventricle ejection fraction ≤45%, a left ventricular end-diastolic diameter ≥3.2 cm/m2 in male and ≥3.3 cm/m2 in female and absence of secondary causes of heart failure such as ischaemic, valvular or hypertensive heart disease. Whole exome sequencing was performed. The genetic variants were compared with the publicly available reference databases Results: Two hundred and eighteen novel damaging variants were identified that have not been previously reported consisting of 85 (84.9%) private variants. Of the 218 novel variants, 33 (15.1%) mutations were recurrent variants i.e. variants occurring in more than one DCM patient. Novel candidate genes for DCM identified included MYL2, MYL7, Kruppel-associated box domain (KRAB) Zinc-fingers gene family, IL23R, FBX022, PLCG1, RyR1, ITGB7 and GNAS Conclusion: The identified novel genetic variants involve multiple gene functions. These imply that DCM is genetically heterogeneous disease that involves the activation of different signalling pathways. The novel variants of candidate genes identified in this study fill the gap and expand the spectrum of genetic landscape in DCM

385. Inhibiting the Myostatin Signaling Pathway using CRISPR/Cas9-Based Repressors

Inhibition of myostatin signaling is a potential method to enhance skeletal muscle growth and has been proposed as a strategy to treat muscle weakness associated with sarcopenia, cachexia, and muscular dystrophies. Current approaches under clinical investigation for inhibiting the myostatin pathway rely on systemic administration of soluble factors, including myostatin blocking antibodies and soluble myostatin receptors. Treatment with these protein-based inhibitors has led to enhanced muscle mass in mouse models of muscular dystrophy and cachexia, but has demonstrated limited efficacy and some adverse side effects in subsequent clinical trials. We hypothesized that a targeted gene regulation strategy to localize inhibition of the myostatin pathway to skeletal muscle fibers may increase the effectiveness and safety of this therapy. The RNA-guided CRISPR/Cas9 system has emerged as a promising platform for targeted gene regulation. Fusion of catalytically inactive, “dead” Cas9 (dCas9) to the Kruppel-associated box (KRAB) domain generates a synthetic repressor capable of highly specific silencing of target genes. dCas9-KRAB repressors can be employed in gene therapy to silence detrimental gene products, repress oncogenes, inhibit viral replication, and treat dominant negative diseases. However, gene delivery of dCas9-KRAB to skeletal muscle in vivo is challenging because the size of the S. pyogenes dCas9 and KRAB domain fusion exceeds the packaging limit of standard AAV vectors. Recently, a smaller Cas9 protein derived from S. aureus was described for AAV delivery and in vivo gene editing. We generated a S. aureus dCas9-KRAB fusion and targeted the myostatin receptor, Acvr2b, for silencing. In cultured mouse myoblasts, S. aureus dCas9-KRAB potently repressed Acvr2b expression by qPCR and resulted in reduced myotube formation following differentiation compared to controls. The S. aureus dCas9-KRAB repressor was packaged into AAV vectors and expressed efficiently in vitro in cultured myoblasts and in vivo following direct injection into the mouse tibialis anterior muscle. Ongoing studies will determine the effects of transcriptional silencing of Acvr2b on myotube diameter in vitro and the efficiency of Acvr2b silencing achieved by engineered dCas9-KRAB repressors in vivo. These studies establish how transcriptional modulation with the CRISPR/Cas9 system can be used to investigate potential therapeutic gene targets for treating neuromuscular disorders.

The B-Subdomain of the Xenopus laevis XFIN KRAB-AB Domain Is Responsible for Its Weaker Transcriptional Repressor Activity Compared to Human ZNF10/Kox1

The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

MAS promoter regulation: a role for Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism

The ACE2 (angiotensin-converting enzyme 2)/Ang-(1-7) [angiotensin-(1-7)]/MAS axis of the RAS (renin-angiotensin system) has emerged as a pathway of interest in treating both cardiovascular disorders and cancer. The MAS protein is known to bind to and be activated by Ang-(1-7); however, the mechanisms of this activation are just starting to be understood. Although there are strong biochemical data regarding the regulation and activation of the AT1R (angiotensin II type1 receptor) and the AT2R (angiotensin II type2 receptor), with models of how AngII (angiotensin II) binds each receptor, fewer studies have characterized MAS. In the present study, we characterize the MAS promoter and provide a potential feedback mechanism that could compensate for MAS degradation following activation by Ang-(1-7). Analysis of ENCODE data for the MAS promoter revealed potential epigenetic control by KRAB (Krüppel-associated box)/KAP-1 (KRAB-associated protein-1). A proximal promoter construct for the MAS gene was repressed by the SOX [SRY (sex-determining region on the Y chromosome) box] proteins SRY, SOX2, SOX3 and SOX14, of which SRY is known to interact with the KRAB domain. The KRAB-KAP-1 complex can be tyrosine-nitrated, causing the dissociation of the KAP-1 protein and thus a potential loss of epigenetic control. Activation of MAS can lead to an increase in nitric oxide, suggesting a feedback mechanism for MAS on its own promoter. The results of the present study provide a more complete view of MAS regulation and, for the first time, suggest biochemical outcomes for nitration of the KRAB domain.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

SummaryHematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.

TRIM28 is required by the mouse KRAB domain protein ZFP568 to control convergent extension and morphogenesis of extra-embryonic tissues

TRIM28 is a transcriptional regulator that is essential for embryonic development and is implicated in a variety of human diseases. The roles of TRIM28 in distinct biological processes are thought to depend on its interaction with factors that determine its DNA target specificity. However, functional evidence linking TRIM28 to specific co-factors is scarce. chatwo, a hypomorphic allele of Trim28, causes embryonic lethality and defects in convergent extension and morphogenesis of extra-embryonic tissues. These phenotypes are remarkably similar to those of mutants in the Krüppel-associated box (KRAB) zinc finger protein ZFP568, providing strong genetic evidence that ZFP568 and TRIM28 control morphogenesis through a common molecular mechanism. We determined that chatwo mutations decrease TRIM28 protein stability and repressive activity, disrupting both ZFP568-dependent and ZFP568-independent roles of TRIM28. These results, together with the analysis of embryos bearing a conditional inactivation of Trim28 in embryonic-derived tissues, revealed that TRIM28 is differentially required by ZFP568 and other factors during the early stages of mouse embryogenesis. In addition to uncovering novel roles of TRIM28 in convergent extension and morphogenesis of extra-embryonic tissues, our characterization of chatwo mutants demonstrates that KRAB domain proteins are essential to determine some of the biological functions of TRIM28.

A Novel Transcriptional Repressor, Rhit, Is Involved in Heat-Inducible and Age-Dependent Expression of Mpv17-Like Protein, a Participant in Reactive Oxygen Species Metabolism

Mpv17-like protein (M-LP) is a protein that has been suggested to be involved in the metabolism of reactive oxygen species. The two M-LP isoforms in mouse, M-LP(S) and M-LP(L), are generated by the alternative usage of promoters. M-LP(S) is expressed exclusively in kidneys after the age of 6 weeks, whereas M-LP(L) is expressed ubiquitously. To elucidate the molecular basis of M-LP(S) expression, we searched for cis-regulatory elements in the promoter region of M-LP(S) and identified heat shock element half-sites as positive elements and a Tramtrack 69K (Ttk 69K) binding site as a negative element. Furthermore, we isolated a novel transcription repressor, Rhit (regulator of heat-induced transcription), that binds to the Ttk 69K binding site within the M-LP(S) promoter by DNA affinity chromatography and confirmed its participation in the transcriptional regulation of M-LP(S) by RNA interference (RNAi). Sequence analysis revealed that Rhit contains a KRAB (Krüppel-associated box) domain and a DNA-binding domain composed of eight C(2)H(2)-type zinc fingers. Interestingly, exposure to heat shock stress resulted in the upregulation of M-LP(S) expression concurrent with the downregulation of Rhit expression. Moreover, the age-dependent expression of M-LP(S) was inversely correlated with that of Rhit. These observations strongly suggest that Rhit acts as a repressor in the heat-induced and age-dependent transcriptional regulation of M-LP(S).

The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

BackgroundExpansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.ResultsHere, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.ConclusionsThe ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.