BTB Domain-containing Protein Research Articles

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68

AbstractRANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

Circ-GTF2I/miR-590-5p Axis Aggravates Myocardial Ischemia-Reperfusion Injury by Regulating Kelch Repeat and BTB Domain-Containing Protein 7.

Purpose We investigated the effect of the circular RNA (circRNA) general transcription factor IIi (GTF2I) on myocardial ischemia (MI) deterioration and neonatal rat cardiomyocyte damage. Methods The cell experiment was performed by using neonatal rat cardiomyocytes. Moreover, a hypoxia/reoxygenation treatment model was established. Cell Counting Kit-8 assay was conducted, and EdU cell proliferation was detected. Cell apoptosis was detected via flow cytometry and quantitative RT-PCR (RT-qPCR). Binding detection was performed through a double-luciferase reporter assay. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) were detected via enzyme-linked immunosorbent assay (ELISA). Results Compared with that in the sham and control groups, circ-GTF2I expression in MIRI and the hypoxia/reoxygenation treatment model was significantly upregulated in vivo and in vitro. The knockdown of circ-GTF2I relieved neonatal rat cardiomyocyte damage and MI. Further detection through the double-luciferase reporter assay confirmed that the binding site of circ-GTF2I to miR-590-5p and miR-590-5p was Kelch repeat and BTB domain-containing protein 7 (KBTBD7). ELISA and RT-qPCR results showed that circ-GTF2I induced the abnormal expressions of IL-6 TNF-α, LDH, Bax, Bcl-2, and Cyt-c in MIRI and the hypoxia/reoxygenation treatment models by regulating miR-590-5p and the heart development transcription factor KBTBD7. Conclusions CircRNA circ-GTF2I aggravated MIRI and neonatal rat cardiomyocyte damage in vivo and in vitro by regulating miR-590-5p and the heart development transcription factor KBTBD7.

Spatial transcriptomics unveils ZBTB11 as a regulator of cardiomyocyte degeneration in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM. Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis. Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.

Genome-Wide Identification and Expression Analysis of the BTB Domain-Containing Protein Gene Family in Sugar Beet

Cercospora leaf spots (CLSs) is a fungal disease of sugar beet caused by C. beticola, which damages leaves and leads to yield cut on sugar beet worldwide. BTB protein genes are critical to plant defense against bacterial infection. Here, 49 members of the BTB protein gene family were identified from the big data of the sugar beet genome, and bioinformatics was used to analyze the BTB protein family. Through molecular techniques, C. beticola of CLS was identified. In addition, the transcriptome data of sugar beet resistant and susceptible materials after C. beticola infection were obtained. Three BTB genes most significantly related to C. beticola stress were screened from the transcriptome data. The three genes are BvBTB1, BvBTB2, and BvBTB3, their full-length cDNA sequences were acquired by RT-PCR. The phenotypes of sugar beet resistant and susceptible materials under different spore concentrations of C. beticola were analyzed. Further, under the stress of C. beticola, qRT-PCR results showed that the expression levels of BvBTB1, BvBTB2, and BvBTB3 in roots and leaves were tissue-specific and expressed differently in various tissues. BvBTB1, BvBTB2, and BvBTB3 were overexpressed in the resistant and susceptible materials within five days after C. beticola infection: the peak appeared on the fifth day, and the highest expression was 25 times that of the control group. However, the lowest was 1.1 times of the control group, moreover, the expression in the resistant material was higher than that in the susceptible material. Overall, these results showed that BvBTB genes were involved in the response in sugar beet to C. beticola infection. Therefore, the study provided a scientific theoretical basis for developing new resistant varieties in sugar beet.

Abstract MP47: Rho-related BTB Domain-containing Protein 1 (RhoBTB1) Attenuates Established Arterial Stiffness In Ang-ii-treated Mice

RhoBTB1 is a transcription target of PPARγ but its function remains unclear. Restoration ofRhoBTB1 by genetic complementation in S-P467L mice expressing dominant negative PPARγmutation reversed hypertension, arterial stiffness, and vascular dysfunction. Here, we asked ifrestoring RhoBTB1 can reverse Ang-II-mediated hypertension and arterial stiffness.To test this hypothesis, S-RhoBTB1 mice, which expressed smooth muscle-specific, tamoxifen(Tx)-inducible RhoBTB1, were implanted with minipumps which released Ang-II (490ng/kg/min, 6weeks) or vehicle. Concurrently, littermate ISM-Cre mice, which expressed smooth muscle-specific, Tx-inducible Cre (but not RhoBTB1), were used as control. Transgenes were induced by5 daily Tx injections 2 weeks after Ang-II pump implantation, and tissue was harvested 2 weeksafter Tx. Measurements were made during a 1 week baseline period, 2 weeks of Ang-II, and 2weeks after transgene activation.Aortic RhoBTB1 expression was decreased in Ang-II-treated ISM-Cre mice (ISM-Cre, Ang-II vs.ISM-Cre, vehicle dCT: 13.6±0.1 vs. 12.4±0.1, n=10), and restored to normal in Ang-II-treated S-RhoBTB1 mice (S-RhoBTB1, Ang-II vs. ISM-Cre, vehicle dCT: 12.8±0.2 vs. 12.4±0.1, n=10-12).Interestingly, pulse-wave velocity, a clinical indicator of arterial stiffness, was significantlydecreased upon RhoBTB1 restoration in Ang-II-treated mice (S-RhoBTB1 vs. ISM-Cre: 2.9±0.1vs. 3.8±0.2, n=22-26). Concomitantly, measures of both pressure-diameter and stress-strainshowed that aortic compliance was improved by RhoBTB1 restoration in Ang-II-treated mice.Interestingly, restoring RhoBTB1 was insufficient to attenuate Ang-II-mediated hypertension (S-RhoBTB1, Ang-II vs. ISM-Cre, Ang-II vs. ISM-Cre, vehicle SBP: 152.1±1.7 vs. 148.0±3.7 vs.115.4± 0.7 mmHg n=12, 8, 6). Currently, we are investigating the mechanism by which RhoBTB1attenuates arterial stiffness.In summary, RhoBTB1 restoration ameliorated Ang-II-mediated arterial stiffness but nothypertension, suggesting that arterial stiffness is not merely a consequence of hypertension butcan be independently regulated from blood pressure. Moreover, there may be novel targets ofRhoBTB1 that can regulate aortic stiffness and be therapeutic.

Abstract P228: RhoBTB1 Is Expressed In Placental Syncytiotrophoblast Cells

Preeclampsia is a pregnancy-specific disorder of hypertension and end-organ dysfunction that poses serious clinical problems for women and their offspring that extend beyond the peripartum period. Abnormal placentation is at the core of preeclampsia. PPARγ-deficient mice exhibit numerous placental defects including malformation of the labyrinth zone, lack of infiltration of fetal blood vessels, and rupture of maternal blood sinuses, suggesting that PPARγ is a critical regulator of placental development and possibly function. Rho-related BTB domain-containing protein 1 (RhoBTB1) is a PPARγ target gene which controls the level of cGMP in vascular smooth muscle cells by functioning as an adaptor delivering phosphodiesterase 5 (PDE5) to the Cullin-3 ubiquitin ligase complex. Analysis of gene expression databases revealed that placenta is among the tissues with the highest level of RhoBTB1 expression. Thus, we examined if the level or cell-specificity of RhoBTB1 expression was altered in preeclampsia. Placental samples were obtained from the Medical College of Wisconsin Maternal Research Placenta and Cord Blood Bank. We examined placentas from term healthy (n=3), preterm non-preeclamptic (n=2), term preeclamptic (n=2) and preterm preeclamptic (n=2) pregnancies. RNAscope in situ hybridization was used to qualitatively evaluate expression of RhoBTB1 and several placental markers. Our results reveal that RhoBTB1 co-localizes with CYP19A1, a gene encoding cytochrome P450 enzyme, a target specific to the syncytiotrophoblast cell layer in the placenta. Expression of RhoBTB1 was not co-localized with platelet-endothelial cell adhesion molecule-1 (PECAM) or with melanoma cell adhesion molecule (MCAM), both endothelial cell markers. These results indicate that RhoBTB1 is specifically expressed in the syncytiotrophoblast cell layer in human placentas. This finding persisted in preterm and preeclamptic placental samples. These data compel us to assess the functional significance of placental RhoBTB1 using conditional knockout and a conditional activatable RhoBTB1 models. Further investigative work is being done to determine if placental RhoBTB1 expression is altered based on ethnic and racial differences.

ZBTB20 is crucial for the specification of a subset of callosal projection neurons and astrocytes in the mammalian neocortex.

Neocortical progenitor cells generate subtypes of excitatory projection neurons in sequential order followed by the generation of astrocytes. The transcription factor zinc finger and BTB domain-containing protein 20 (ZBTB20) has been implicated in regulation of cell specification during neocortical development. Here, we show that ZBTB20 instructs the generation of a subset of callosal projections neurons in cortical layers II/III in mouse. Conditional deletion of Zbtb20 in cortical progenitors, and to a lesser degree in differentiating neurons, leads to an increase in the number of layer IV neurons at the expense of layer II/III neurons. Astrogliogenesis is also affected in the mutants with an increase in the number of a specific subset of astrocytes expressing GFAP. Astrogliogenesis is more severely disrupted by a ZBTB20 protein containing dominant mutations linked to Primrose syndrome, suggesting that ZBTB20 acts in concert with other ZBTB proteins that were also affected by the dominant-negative protein to instruct astrogliogenesis. Overall, our data suggest that ZBTB20 acts both in progenitors and in postmitotic cells to regulate cell fate specification in the mammalian neocortex.

Dysfunction of Cullin 3 RING E3 ubiquitin ligase causes vasoconstriction and increased sodium reabsorption in diabetes

Impaired endothelium-mediated vasodilation and/or increased sensitivity to vasoconstrictors lead to vascular smooth muscle cell (VSMC) dysfunction in individuals with diabetes. Diabetic nephropathy is associated with a considerably higher risk of cardiovascular disease and death than their nondiabetic counterparts. We studied the activity of Cullin 3 RING ubiquitin ligase (CRL3) and its substrates in mice using an intraperitoneal injection of streptozotocin (STZ) and db/db mice. The levels of CRL3 adaptors, including Kelch-like 2/3 (KLHL2/3) and Rho-related BTB domain-containing protein 1, were significantly decreased in the aortic tissues and heart of the STZ group, whereas the levels of Cullin 3 (CUL3) and its neddylated derivatives were substantially increased. Decreased KLHL3 expression and significantly increased expression of NEDD8 conjugates were observed in the kidneys of db/db mice. The neddylation inhibitor MLN4924 decreased the degradation of KLHL2/KLHL3 under high-glucose conditions with/without insulin, and transfection with KLHL2 promoted the degradation of its substrates with-no-lysine (WNK) kinases. Increased abundance of WNK3, RhoA/ROCK activity and phosphodiesterase 5 enhanced the sensibility to vasoconstrictors and impaired vasodilation. Moreover, WNK3 localized in VSMCs undergoing cell division, and high-glucose medium increased WNK3 signaling in VSMCs undergoing mitosis, which might explain the increased thickness of aortic tissues in subjects with diabetes. Increases in WNK4 abundance resulted in increased sodium reabsorption in the distal renal tubules. Thus, KLHL2/RhoBTB1/KLHL3 inactivation in the aortic tissues and kidney is a result of excessive activation of neddylation in hyperglycemia and hyperinsulinemia, which affects vascular tone and sodium reabsorption.

Zinc finger and BTB domain-containing protein 20 aggravates angiotensin II-induced cardiac remodeling via the EGFR-AKT pathway

Zinc finger and BTB domain-containing protein 20 (ZBTB20) play an important role in glucose and lipid homeostasis. ZBTB20 was shown to be a crucial protein for the maintenance of cardiac contractile function. However, the role of ZBTB20 in cardiac response remodeling has not been elucidated. Thus, this study aimed to explore the role of ZBTB20 in cardiac remodeling following angiotensin II insult. Mice were subjected to angiotensin II infusion to induce a cardiac adverse remodeling model. An adeno-associated virus (AAV) 9 system was used to deliver ZBTB20 to the mouse heart. Here, we demonstrate that ZBTB20 expression is elevated in angiotensin II-induced cardiac remodeling and in response to cardiomyocyte insults. Furthermore, AAV9-mediated overexpression of ZBTB20 caused cardiac wall hypertrophy, chamber dilation, increased fibrosis, and reduced ejection fraction. Additionally, ZBTB20 siRNA protected cardiomyocytes from angiotensin II-induced hypertrophy. Mechanistically, ZBTB20 interferes with EGFR and Akt signaling and modulates the remodeling response. Overexpression of constitutively active Akt counteracts ZBTB20 knockdown-mediated protection of adverse cardiac remodeling. These findings illustrate the role of ZBTB20 in the transition of adverse cardiac remodeling toward heart failure and provide evidence for the molecular programs inducing adverse cardiac remodeling. KEY MESSAGES: ZBTB20 is a transcription factor from the POK family. ZBTB20 is upregulated in heart tissue treated with angiotensin II. ZBTB20 influences cardiomyocyte hypertrophy via the EGFR-Akt pathway. Akt continuous activation leads to similar results to ZBTB20 overexpression.

Epigenetic silencing of ZBTB28 promotes renal cell carcinogenesis.

Zinc finger and BTB domain-containing protein 28 (ZBTB28) is a potential tumor suppressor for some cancers. However, its epigenetic regulation and functions in renal cell carcinoma (RCC) remain to be elucidated. The expression of ZBTB28 mRNA was analyzed by semi-quantitative reverse transcription polymerase chain reaction (PCR) in nine RCC cell lines and normal kidney tissues. Methylation status of ZBTB28 promoter was assessed by methylation-specific PCR in RCC cell lines, primary RCC, tumors and adjacent tissues. The involvement of ZBTB28 in cell proliferation and migration was investigated. ZBTB28 promoter was hypermethylated in 88.9% (8/9) of RCC cell lines with reduced ZBTB28 mRNA expression, and could be reversed by DNA methyltransferase inhibitors. The methylation of ZBTB28 promoter was detected in 73.5% (36/49) of primary RCC tissues, compared with 7.1% (1/14) in normal tissues. Overexpression of ZBTB28 significantly inhibited RCC cell proliferation and migration, and induced apoptosis. Further analyses revealed that ZBTB28 upregulation could inhibit multiple oncogenic signaling transduction pathways. ZBTB28 is frequently silenced by promoter methylation in RCC pathogenesis and functions as a novel tumor suppressive gene. ZBTB28 may be a potential target for the development of RCC therapeutic strategies.

MiR-216a-5p Suppresses the Proliferation, Invasion and Migration of Fibroblast-Like Synoviocyte in Rheumatoid Arthritis by Targeting Zinc Finger and BTB Domain-Containing Protein 2 (ZBTB2)

MicroRNAs have been reported to be associated with the initiation and progression of rheumatoid arthritis (RA). miR-216a-5p, one of the miRNAs, is involved in cancer cell proliferation, invasion and migration. However, the role of miR-216a-5p in RA remains to be explored. The expressions of miR-216a-5p and zinc finger and BTB domain-containing protein 2 (ZBTB2) in fibroblast-like synoviocytes (FLS) of RA or healthy controls were detected by qRT-PCR and western blot analysis. Transfection of overexpressed and silenced miR-216a-5p were performed to explore the functional role of miR-216a-5p in RA-FLS. Cell Counting Kit-8 (CCK-8) assay and transwell assay were employed to assess cell proliferation and cell invasion, respectively. Moreover, luciferase reporter assay was executed to verify the combination of miR-216a-5p and ZBTB2. The results showed that miR-216a-5p expression in RA-FLS was downregulated than healthy controls. Overexpres-sion of miR-216a-5p inhibited RA-FLS cell proliferation, invasion and migration, while miR-216a-5p silencing revealed the opposite results. In addition, ZBTB2 was identified to be a direct target of miR-216a-5p in RA-FLS and its expression was higher than that in healthy controls. Rescue experiments revealed that ZBTB2 overexpression reversed the effects of miR-216a-5p on the proliferation, invasion and migration of RA-FLS. These data indicated the suppressive role of miR-216a-5p in RA-FLS via the regulation of ZBTB2, suggesting that miR-216a-5p and ZBTB2 may be the new targets for the treatment of RA.

Reversal of Arterial Stiffness by Rho‐related BTB Domain‐containing Protein 1 (RhoBTB1)

Rho-related BTB domain-containing protein (RhoBTB1) is a novel transcriptional target of peroxisome proliferator activated receptor Gamma (PPARg). The physiological and molecular function of RhoBTB1 is poorly understood. Human individuals carrying PPARg dominant negative mutation P467L develop early onset hypertension. Mice expressing smooth muscle-specific P467L also develop hypertension, vascular dysfunction, and arterial stiffness. Interestingly, P467L-mediated hypertension and its sequelae can be reversed by restoration of RhoBTB1 in smooth muscle, suggesting that RhoBTB1 mediates the anti-hypertension effect of PPARg in vascular smooth muscle cells. We recently found that RhoBTB1 preserves the nitric oxide-cGMP pathway by serving as a substrate adaptor for Cullin-3 and promoting phosphodiesterase 5 ubiquitination. Our current study explores the role of RhoBTB1 in angiotensin II (Ang-II) induced hypertension, a model that represents a broader hypertension population. We hypothesize that RhoBTB1 transgene expression in vascular smooth muscle will attenuate Ang-II-mediated hypertension and its sequelae. Mice expressing smooth muscle-specific, Tamoxifen inducible RhoBTB1 transgene (S-RhoBTB1) were used as experimental animals, whereas mice expressing smooth muscle-specific, Tamoxifen inducible Cre-recombinase (ISM-Cre) were used as control. Mice received 6-weeks of Ang-II infusion via osmotic mini pump at 490ng/kg/min. Transgene was induced by five daily Tamoxifen injections two weeks after Ang-II pumps implantation, allowing hypertension to be established before transgene activation. Blood pressure was monitored by radiotelemetry. Systolic, diastolic, and mean arterial blood pressure was significantly increased in response to Ang-II. RhoBTB1 transgene expression was not sufficient to reverse the high blood pressure. We also tested vascular reactivity in conduit and resistant arteries using wire myography. Ang-II infusion significantly impaired the vasodilation response to acetylcholine in carotid and mesenteric arteries, suggesting a defect in endothelium-mediated vasodilation. Like blood pressure, RhoBTB1 transgene expression was not sufficient to reverse the vasodilation impairment. Finally, we measured arterial stiffness using pulse-wave velocity (PWV). Both ISM-Cre and S-RhoBTB1 mice showed increased arterial stiffness two weeks after Ang-II infusion, indicated by a higher PWV. PWV in S-RhoBTB1 mice started to decline one-week post Tamoxifen and became significantly lowered (nearly reversed) two weeks after Tamoxifen. In comparison, PWV in ISM-Cre mice remained high till the end of the 6-weeks protocol. To explore the underlying mechanism, we evaluated the aortic compliance using pressure myograph in the absence of vascular tone and reactivity. Both pressure-diameter and stress-strain relationship indicate that RhoBTB1 transgene expression in smooth muscle improved the compliance of aorta in the presence of Ang-II, suggesting RhoBTB1 might attenuate arterial stiffness by acting on the extracellular matrix. Our study revealed a novel protective effect of RhoBTB1: attenuating established Ang-II-induced arterial stiffness.

Single nucleotide variations in ZBTB46 are associated with post-thrombolytic parenchymal haematoma.

Haemorrhagic transformation is a complication of recombinant tissue-plasminogen activator treatment. The most severe form, parenchymal haematoma, can result in neurological deterioration, disability, and death. Our objective was to identify single nucleotide variations associated with a risk of parenchymal haematoma following thrombolytic therapy in patients with acute ischaemic stroke. A fixed-effect genome-wide meta-analysis was performed combining two-stage genome-wide association studies (n = 1904). The discovery stage (three cohorts) comprised 1324 ischaemic stroke individuals, 5.4% of whom had a parenchymal haematoma. Genetic variants yielding a P-value < 0.05 1 × 10-5 were analysed in the validation stage (six cohorts), formed by 580 ischaemic stroke patients with 12.1% haemorrhagic events. All participants received recombinant tissue-plasminogen activator; cases were parenchymal haematoma type 1 or 2 as defined by the European Cooperative Acute Stroke Study (ECASS) criteria. Genome-wide significant findings (P < 5 × 10-8) were characterized by in silico functional annotation, gene expression, and DNA regulatory elements. We analysed 7 989 272 single nucleotide polymorphisms and identified a genome-wide association locus on chromosome 20 in the discovery cohort; functional annotation indicated that the ZBTB46 gene was driving the association for chromosome 20. The top single nucleotide polymorphism was rs76484331 in the ZBTB46 gene [P = 2.49 × 10-8; odds ratio (OR): 11.21; 95% confidence interval (CI): 4.82-26.55]. In the replication cohort (n = 580), the rs76484331 polymorphism was associated with parenchymal haematoma (P = 0.01), and the overall association after meta-analysis increased (P = 1.61 × 10-8; OR: 5.84; 95% CI: 3.16-10.76). ZBTB46 codes the zinc finger and BTB domain-containing protein 46 that acts as a transcription factor. In silico studies indicated that ZBTB46 is expressed in brain tissue by neurons and endothelial cells. Moreover, rs76484331 interacts with the promoter sites located at 20q13. In conclusion, we identified single nucleotide variants in the ZBTB46 gene associated with a higher risk of parenchymal haematoma following recombinant tissue-plasminogen activator treatment.

Elevated expression of sepiapterin reductase, regulator of G protein signaling 1, hypothetical protein CXorf58 homolog, and zinc finger and BTB domain-containing protein 21 isoform X2 is associated with progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers associated with high mortality rate. Understanding of events leading to HCC pathophysiology is essential for its better management. We earlier reported development of a novel rodent model by administrating chemical carcinogens, DEN, and 2-AAF for study of HCC at very early stage. 2D-Electrophoresis analysis of total serum proteins identified several differentially expressed proteins in animals undergoing tumorigenesis. MALDI-TOF-MS/MS analyses were performed to characterize the differentially expressed proteins. Further real-time PCR analyses were taken place to quantify the transcript expression for the identified proteins at HCC initiation and tumor stages. Considering protein-protein interactions among the experimentally identified proteins and their interacting neighbors, a protein network has been analyzed that provided further insight into molecular events taking place during HCC development. Histological changes confirmed HCC initiation and hepatotumorigenesis at 1 and 4 months post carcinogen treatment, respectively. Four differentially expressed proteins were identified which were further characterized as regulator of G protein signaling 1 (RGS1), sepiapterin reductase (SPR), similar to zinc finger and BTB domain-containing protein 21 isoform X2 (ZNF295), and a hypothetical protein CXorf58 homolog. Quantification of transcripts for these proteins revealed elevation in their expression both at initiation and tumorigenesis stages. The study deciphers the regulatory role of these proteins during HCC progression.

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells.

Double sex and mab-3-related transcription factor 1 (Dmrt1), which is expressed in goat male germline stem cells (mGSCs) and Sertoli cells, is one of the most conserved transcription factors involved in sex determination. In this study, we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs. Dmrt1 recruited promyelocytic leukemia zinc finger (Plzf), also known as zinc finger and BTB domain-containing protein 16 (Zbtb16), to repress the Toll-like receptor 4 (TLR4)-dependent inflammatory signaling pathway and nuclear factor (NF)-κB. Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells, while the expression of proinflammatory factors were significantly enhanced. We also demonstrated that Dmrt1 stimulated proliferation of mGSCs, but repressed apoptosis caused by the immune response. Thus, Dmrt1 is sufficient to reduce inflammation in the testes, thereby establishing the stability of spermatogenesis and the testicular microenvironment.

Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca-The Blue Mussel (Mytilus edulis), Soft Shell Clam (Mya arenaria), Eastern Oyster (Crassostrea virginica) and Atlantic Jacknife Clam (Ensis leei).

Simple SummaryOysters and clams form an important component of the food chain and food security and are of considerable commercial value worldwide. They are affected by pollution and climate change, as well as a range of infections, some of which are opportunistic. For aquaculture purposes they are furthermore of great commercial value and changes in their immune responses can also serve as indicators of changes in ocean environments. Therefore, studies into understanding new factors in their immune systems may aid new biomarker discovery and are of considerable value. This study assessed new biomarkers relating to changes in protein function in four economically important marine molluscs, the blue mussel, soft shell clam, Eastern oyster, and Atlantic jacknife clam. These findings indicate novel regulatory mechanisms of important metabolic and immunology related pathways in these mollusks. The findings provide new understanding to how these pathways function in diverse ways in different animal species as well as aiding new biomarker discovery for Mollusca aquaculture.Oysters and clams are important for food security and of commercial value worldwide. They are affected by anthropogenic changes and opportunistic pathogens and can be indicators of changes in ocean environments. Therefore, studies into biomarker discovery are of considerable value. This study aimed at assessing extracellular vesicle (EV) signatures and post-translational protein deimination profiles of hemolymph from four commercially valuable Mollusca species, the blue mussel (Mytilus edulis), soft shell clam (Mya arenaria), Eastern oyster (Crassostrea virginica), and Atlantic jacknife clam (Ensis leei). EVs form part of cellular communication by transporting protein and genetic cargo and play roles in immunity and host–pathogen interactions. Protein deimination is a post-translational modification caused by peptidylarginine deiminases (PADs), and can facilitate protein moonlighting in health and disease. The current study identified hemolymph-EV profiles in the four Mollusca species, revealing some species differences. Deiminated protein candidates differed in hemolymph between the species, with some common targets between all four species (e.g., histone H3 and H4, actin, and GAPDH), while other hits were species-specific; in blue mussel these included heavy metal binding protein, heat shock proteins 60 and 90, 2-phospho-D-glycerate hydrolyase, GTP cyclohydrolase feedback regulatory protein, sodium/potassium-transporting ATPase, and fibrinogen domain containing protein. In soft shell clam specific deimination hits included dynein, MCM3-associated protein, and SCRN. In Eastern oyster specific deimination hits included muscle LIM protein, beta-1,3-glucan-binding protein, myosin heavy chain, thaumatin-like protein, vWFA domain-containing protein, BTB domain-containing protein, amylase, and beta-catenin. Deiminated proteins specific to Atlantic jackknife clam included nacre c1q domain-containing protein and PDZ domain-containing protein In addition, some proteins were common as deiminated targets between two or three of the Bivalvia species under study (e.g., EP protein, C1q domain containing protein, histone H2B, tubulin, elongation factor 1-alpha, dominin, extracellular superoxide dismutase). Protein interaction network analysis for the deiminated protein hits revealed major pathways relevant for immunity and metabolism, providing novel insights into post-translational regulation via deimination. The study contributes to EV characterization in diverse taxa and understanding of roles for PAD-mediated regulation of immune and metabolic pathways throughout phylogeny.

Downregulation of the ubiquitin ligase KBTBD8 prevented epithelial ovarian cancer progression

ObjectivesKelch repeat and BTB domain-containing protein 8, KBTBD8, has been identified as a female fertility factor. However, there have been no reports on the role of KBTBD8 in the progression of epithelial ovarian cancer, EOC. Our study aimed to address this issue.MethodsWe first examine KBTBD8 expression in EOC tissues and cells. Next, we performed RNA sequencing to reveal the overall mechanism. Then we investigated the roles of KBTBD8 in the proliferation, migration, and health status of cultured EOC cells. Finally, we employed tumor xenograft models to evaluate the role of KBTBD8 in vivo.ResultsFirst, KBTBD8 level was significantly higher in EOC tissues and cells. Next, comparative RNA sequencing identified more tumorigenesis-related genes that KBTBD8 might regulate. Then we found that KBTBD8 knockdown significantly decreased EOC cell proliferation, migration, and the activities of multiple tumorigenesis-related kinases. Finally, KBTBD8 knockdown significantly diminished ovarian tumor formation in vivo.ConclusionProper KBTBD8 level is essential for the healthy growth of ovarian somatic cells, such as ovarian epithelial cells. Excessive KBTBD8 might be a significant impetus for EOC progression. KBTBD8 reduction greatly inhibits EOC proliferation and migration.

Zbtb7b suppresses aseptic inflammation by regulating m6A modification of IL6 mRNA

Radiotherapy is a crucial approach for treating tumors. However, radiation-induced aseptic inflammation is a common complication. Radiation pneumonitis is the acute manifestation of radiation-induced lung disease, and interleukin 6 (IL-6) is a major proinflammatory cytokine involved in radiation-induced lung injury. Here we found that silencing Zinc finger and BTB domain-containing protein 7B (Zbtb7b) resulted in higher radiation-induced IL-6 production in THP1 cells and BEAS-2B lung bronchial epithelial cells. Mechanistically, Zbtb7b recruited RNA demethylase ALKBH5 to IL6 mRNA. Subsequentially, it demethylated N6-methyladenosine (m6A) modification of IL6 mRNA and inhibited its nuclear export. Thus, Zbtb2b epigenetically suppresses irradiation-induced IL-6 production in the lungs via inhibiting the m6A modification and nucleocytoplasmic transport of IL6 mRNA, serving as a new potential predictive marker and therapeutic target in radiation pneumonitis treatment.

Zinc finger and BTB domain-containing protein 46 is essential for survival and proliferation of acute myeloid leukemia cell line but dispensable for normal hematopoiesis.

Zinc finger and BTB domain-containing protein 46 (Zbtb46) is a transcription factor identified in classical dendritic cells, and maintains dendritic cell quiescence in a steady state. Zbtb46 has been reported to be a negative indicator of acute myeloid leukemia (AML). We found that Zbtb46 was expressed at a relatively higher level in hematopoietic stem and progenitor cells (HSPCs) compared to mature cells, and higher in AML cells compared to normal bone marrow (BM) cells. However, the role of Zbtb46 in HSPCs and AML cells remains unclear. Therefore, we sought to elucidate the effect of Zbtb46 in normal hematopoiesis and AML cells. We generated Zbtb46 and Zbtb46Mx1-Cre mice. The deletion of Zbtb46 in Zbtb46Mx1-Cre mice was induced by intraperitoneal injection of double-stranded poly (I). poly (C) (poly(I:C)), and referred as Zbtb46 cKO. After confirming the deletion of Zbtb46, the frequency and numbers of HSPCs and mature blood cells were analyzed by flow cytometry. Serial intraperitoneal injection of 5-fluorouracil was administrated to determine the repopulation ability of HSCs from Zbtb46 and Zbtb46 cKO mice. The correlation between Zbtb46 expression and prognosis was analyzed using the data from the Cancer Genome Atlas. To investigate the role of Zbtb46 in AML cells, we knocked down the expression of Zbtb46 in THP-1 cells using lentiviral vectors expressing small hairpin RNAs targeting Zbtb46. Cell proliferation rate was determined by cell count assay. Cell apoptosis and bromodeoxyuridine incorporation were determined by flow cytometry. The percentages and absolute numbers of HSPCs and mature blood cells were comparable in Zbtb46 cKO mice and its Zbtb46 littermates (Zbtb46vs. Zbtb46 cKO, HPC: 801,310 ± 84,282 vs. 907,202 ± 97,403, t = 0.82, P = 0.46; LSK: 86,895 ± 7802 vs. 102,210 ± 5025, t = 1.65, P = 0.17; HSC: 19,753 ± 3116 vs. 17,608 ± 3508, t = 0.46, P = 0.67). The repopulation ability of HSCs from Zbtb46Mx1-Cre mice was similar to those from Zbtb46 control (P = 0.26). Zbtb46 had elevated expression in AML cells compared to total BM cells from normal control. Knockdown of Zbtb46 in THP-1 cells led to a significant increase in cell apoptosis and reduced cell growth and proliferation. Collectively, our data indicate that Zbtb46 is essential for survival and proliferation of AML cells, but dispensable for normal hematopoiesis.