Suresh B. Pakala

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (opd) Gene Cluster Found in Flavobacterium sp.

ABSTRACT Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved. Two opd plasmids, pPDL2 from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase. Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF. We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion. The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.

E3 ubiquitin ligase COP1 regulates the stability and functions of MTA1.

Metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is widely upregulated in human cancers. However, the mechanism for regulating its protein stability remains unknown. Here we report that MTA1 is an ubiquitinated protein and targeted by the RING-finger E3 ubiquitin–protein ligase constitutive photomorphogenesis protein 1 (COP1) for degradation via the ubiquitin–proteasome pathway. Induced expression of wild-type COP1 but not its RING motif mutants promotes the ubiquitination and degradation of MTA1, indicating that the ligase activity is required for the COP1-mediated proteolysis of MTA1. Conversely, depletion of endogenous COP1 resulted in a marked decrease in MTA1 ubiquitination, accompanied by a pronounced accumulation of MTA1 protein. MTA1, in turn, destabilizes COP1 by promoting its autoubiquitination, thus creating a tight feedback loop that regulates both MTA1 and COP1 protein stability. Accordingly, disruption of the COP1-mediated proteolysis by ionizing radiation leads to MTA1 stabilization, accompanied by an increased coregulatory function of MTA1 on its target. Furthermore, we discovered that MTA1 is required for optimum DNA double-strand break repair after ionizing radiation. These findings provide novel insights into the regulation of MTA1 protein and reveal a novel function of MTA1 in DNA damage response.

TGF-β1 signaling targets metastasis-associated protein 1, a new effector in epithelial cells

In spite of a large number of transforming growth factor β1 (TGF-β1)-regulated genes, the nature of its targets with roles in transformation continues to be poorly understood. Here, we discovered that TGF-β1 stimulates transcription of metastasis-associated protein 1 (MTA1), a dual master coregulator, in epithelial cells, and that MTA1 status is a determinant of TGF-β1-induced epithelial-to-mesenchymal transition (EMT) phenotypes. In addition, we found that MTA1/polymerase II/activator protein-1 (AP-1) co-activator complex interacts with the FosB-gene chromatin and stimulates its transcription, and FosB in turn, utilizes FosB/histone deacetylase 2 complex to repress E-cadherin expression in TGF-β1-stimulated mammary epithelial cells. These findings suggest that TGF-β1 regulates the components of EMT via stimulating the expression of MTA1, which in turn, induces FosB to repress E-cadherin expression and thus, revealed an inherent function of MTA1 as a target and effector of TGF-β1 signaling in epithelial cells.

Transcriptomic landscape of breast cancers through mRNA sequencing

Breast cancer is a heterogeneous disease with a poorly defined genetic landscape, which poses a major challenge in diagnosis and treatment. By massively parallel mRNA sequencing, we obtained 1.2 billion reads from 17 individual human tissues belonging to TNBC, Non-TNBC, and HER2-positive breast cancers and defined their comprehensive digital transcriptome for the first time. Surprisingly, we identified a high number of novel and unannotated transcripts, revealing the global breast cancer transcriptomic adaptations. Comparative transcriptomic analyses elucidated differentially expressed transcripts between the three breast cancer groups, identifying several new modulators of breast cancer. Our study also identified common transcriptional regulatory elements, such as highly abundant primary transcripts, including osteonectin, RACK1, calnexin, calreticulin, FTL, and B2M, and “genomic hotspots” enriched in primary transcripts between the three groups. Thus, our study opens previously unexplored niches that could enable a better understanding of the disease and the development of potential intervention strategies.

Revelation of p53-independent function of MTA1 in DNA damage response via modulation of the p21 WAF1-proliferating cell nuclear antigen pathway.

Although metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, is a DNA-damage response protein and regulates p53-dependent DNA repair, it remains unknown whether MTA1 also participates in p53-independent DNA damage response. Here, we provide evidence that MTA1 is a p53-independent transcriptional corepressor of p21WAF1, and the underlying mechanism involves recruitment of MTA1-histone deacetylase 2 (HDAC2) complexes onto two selective regions of the p21WAF1 promoter. Accordingly, MTA1 depletion, despite its effect on p53 down-regulation, superinduces p21WAF1, increases p21WAF1 binding to proliferating cell nuclear antigen (PCNA), and decreases the nuclear accumulation of PCNA in response to ionizing radiation. In support of a p53-independent role of MTA1 in DNA damage response, we further demonstrate that induced expression of MTA1 in p53-null cells inhibits p21WAF1 promoter activity and p21WAF1 binding to PCNA. Consequently, MTA1 expression in p53-null cells results in increased induction of γH2AX foci and DNA double strand break repair, and decreased DNA damage sensitivity following ionizing radiation treatment. These findings uncover a new target of MTA1 and the existence of an additional p53-independent role of MTA1 in DNA damage response, at least in part, by modulating the p21WAF1-PCNA pathway, and thus, linking two previously unconnected NuRD complex and DNA-damage response pathways.

Regulation of NF-κB Circuitry by a Component of the Nucleosome Remodeling and Deacetylase Complex Controls Inflammatory Response Homeostasis

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-κB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-κB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1·HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1−/− mice were high compared with wild-type mice. Accordingly, MTA1−/− mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-κB circuitry, in the regulation of inflammatory responses.

Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

In addition to its function as an Arp2/3 complex subunit, Arp1cb interacts with and stimulates Aurora A at centrosomes, functioning in cell cycle progression.

MORC2 Signaling Integrates Phosphorylation-Dependent, ATPase-Coupled Chromatin Remodeling during the DNA Damage Response

Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.

Carcinoembryonic Antigen Interacts with TGF-β Receptor and Inhibits TGF-β Signaling in Colorectal Cancers

As a tumor marker for colorectal cancers, carcinoembryonic antigen (CEA) enhances the metastatic potential of cancer cells. CEA functions as an intercellular adhesion molecule and is upregulated in a wide variety of human cancers. However, the molecular mechanisms by which CEA mediates metastasis remain to be understood. Transforming growth factor-β (TGF-β) signaling regulates both tumor suppression and metastasis, and also contributes to the stimulation of CEA transcription and secretion in colorectal cancer cells. However, it remains unknown whether CEA, in turn, influences TGF-β functions and if a regulatory cross-talk exists between CEA and the TGF-β signaling pathway. Here, we report that CEA directly interacts with TGF-β receptor and inhibits TGF-β signaling. Targeting CEA with either CEA-specific antibody or siRNA rescues TGF-β response in colorectal cancer cell lines with elevated CEA, thereby restoring the inhibitory effects of TGF-β signaling on proliferation. CEA also enhances the survival of colorectal cancer cells in both local colonization and liver metastasis in animal study. Our study provides novel insights into the interaction between CEA and TGF-β signaling pathway and establishes a negative feedback loop in amplifying the progression of colon cancer cells to more invasive phenotypes. These findings offer new therapeutic opportunities to inhibit colorectal cancer cell proliferation by cotargeting CEA in promoting tumor-inhibitory action of the TGF-β pathway.

MTA1 promotes STAT3 transcription and pulmonary metastasis in breast cancer

Overexpression of the prometastatic chromatin modifier protein metastasis tumor antigen 1 (MTA1) in human cancer contributes to tumor aggressiveness, but the role of endogenous MTA1 in cancer has not been explored. Here, we report the effects of selective genetic depletion of MTA1 in a physiologically relevant spontaneous mouse model of breast cancer pulmonary metastasis. We found that MTA1 acts as a mandatory modifier of breast-to-lung metastasis without effects on primary tumor formation. The underlying mechanism involved MTA1-dependent stimulation of STAT3 transcription through action on the MTA1/STAT3/Pol II coactivator complex, and, in turn, on the expression and functions of STAT3 target genes including Twist1. Accordingly, we documented a positive correlation between levels of MTA1 and STAT3 in publicly available breast cancer data sets. Together, our findings reveal an essential modifying role of the physiologic level of MTA1 in supporting pulmonary metastasis of breast cancer.