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As a typical member of intrinsically disordered proteins (IDPs), HMGA1a carries many post-translational modifications (PTMs). To study the undefined function of acidic tail phosphorylations, seven HMGA1a proteins with site-specific modification(s) were chemically synthesized via Ser/Thr ligation. We found that the phosphorylations significantly inhibit HMGA1a-P53 interaction and the phosphorylations can induce conformational change of HMGA1a from an "open state" to a "close state." Notably, the positively charged lysine-arginine (KR) clusters are responsible for modulating HMGA1a conformation via electrostatic interaction with the phosphorylated acidic tail. Finally, we used a synthetic protein-affinity purification mass spectrometry (SP-AP-MS) methodology to profile the specific interactors, which further supported the function of HMGA1a phosphorylation. Collectively, this study highlights a mechanism for regulating IDPs' conformation and function by phosphorylation of non-protein-binding domain and showcases that the protein chemical synthesis in combination with mass spectrometry can serve as an efficient tool to study the IDPs' PTMs.
Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).
The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.
High Mobility Group Protein A1a (HMGA1a) is a highly abundant nuclear protein, which plays a crucial role during embryogenesis, cell differentiation, and neoplasia. Here, we present the first ever NMR-based structural ensemble of full length HMGA1a. Our results show that the protein is not completely random coil but adopts a compact structure consisting of transient long-range contacts, which is regulated by post-translational phosphorylation. The CK2-, cdc2- and cdc2/CK2-phosphorylated forms of HMGA1a each exhibit a different binding affinity towards the PRD2 element of the NFκB promoter. Our study identifies connected regions between phosphorylation sites in the wildtype ensemble that change considerably upon phosphorylation, indicating that these posttranslational modifications sites are part of an electrostatic contact network that alters the structural ensemble by shifting the conformational equilibrium. Moreover, ITC data reveal that the CK2-phosphorylated HMGA1a exhibits a different DNA promoter binding affinity for the PRD2 element. Furthermore, we present the first structural model for AT-hook 1 of HMGA1a that can adopt a transient α-helical structure, which might serve as an additional regulatory mechanism in HMAG1a. Our findings will help to develop new therapeutic strategies against HMGA1a-associated cancers by taking posttranslational modifications into consideration.
Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells. Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2'-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5' splice site of exon 1. Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5' splice site. Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5' splice site of exon 1 offers novel insight on 5' splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved.
High-mobility group AT-hook 1 (HMGA1) protein is an important nuclear factor that activates gene transcription by binding to AT-rich sequences in the promoter region of DNA. We previously demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR) gene and individuals with defects in HMGA1 have decreased INSR expression and increased susceptibility to type 2 diabetes mellitus. In addition, there is evidence that intracellular regulatory molecules that are employed by the INSR signaling system are involved in post-translational modifications of HMGA1, including protein phosphorylation. It is known that phosphorylation of HMGA1 reduces DNA-binding affinity and transcriptional activation. In the present study, we investigated whether activation of the INSR by insulin affected HMGA1 protein phosphorylation and its regulation of gene transcription. Collectively, our findings indicate that HMGA1 is a novel downstream target of the INSR signaling pathway, thus representing a new critical nuclear mediator of insulin action and function.
The forkhead transcription factor (FoxO1) is a master transcriptional regulator of fundamental cellular processes ranging from cell proliferation and differentiation to inflammation and metabolism. However, despite its relevance, the mechanism(s) underlying FoxO1 gene regulation are largely unknown. We have previously shown that the chromatin factor high-mobility group A1 (HMGA1) plays a key role in the transcriptional regulation of glucose-responsive genes, including some that are involved in FoxO1-mediated glucose metabolism. Here we investigated the impact of HMGA1 on FoxO1 gene expression.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation.
Although pancreatic ductal adenocarcinoma is a common and almost uniformly fatal cancer, little is known about the molecular events that lead to tumor progression. The high-mobility group A1 (HMGA1) protein is an architectural transcription factor that has been implicated in the pathogenesis and progression of diverse human cancers, including pancreatic ductal adenocarcinoma. In this study, we investigated HMGA1 expression in pancreatic ductal adenocarcinoma cell lines and surgically resected tumors to determine whether it could be a marker for more advanced disease. By real-time quantitative RT-PCR, we measured HMGA1a mRNA in cultured pancreatic ductal adenocarcinoma cell lines and found increased levels in all cancer cells compared with normal pancreatic tissue. To investigate HMGA1 in primary human tumors, we performed immunohistochemical analysis of 125 cases of pancreatic adenocarcinoma and 99 precursor lesions (PanIN 1-3). We found nuclear staining for HMGA1 in 98% of cases of pancreatic adenocarcinoma, but only 43% of cases of PanIN precursor lesions. Moreover, HMGA1 immunoreactivity correlates positively with decreased survival and advanced tumor and PanIN grade. These results suggest that HMGA1 promotes tumor progression in pancreatic ductal adenocarcinoma and could be a useful biomarker and rational therapeutic target in advanced disease.
Cellular senescence is a state of terminal proliferative arrest that plays key roles in aging by preventing stem cell renewal and by inducing the expression of a series of inflammatory factors including many secreted proteins with paracrine effects. The in vivo identification of senescent cells is difficult due to the absence of universal biomarkers. Chromatin modifications are key aspects of the senescence transition and may provide novel biomarkers. We used a combined protein profiling and bottom-up mass spectrometry approach to characterize the isoforms and post-translational modifications of chromatin proteins over time in post-mitotic human fibroblasts in vitro. We show that the H2B type 1-K variant is specifically enriched in deep senescent cells with persistent DNA damage. This accumulation was not observed in quiescent cells or in cells induced into senescence without DNA damage by expression of the RAF kinase. Similarly, HMGA1a di-methylated and HMGA1b tri-phosphorylated forms accumulated exclusively in the chromatin of cells in deep senescent conditions with persistent DNA damage. H2B type 1-K and modified HMGA1 may thus represent novel biomarkers of senescent cells containing persistent DNA damage.
Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism.
As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.
The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact.
PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor-positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found a frequent allelic expression imbalance between the missense mutant and wild-type PIK3CA alleles in breast tumors from the METABRIC (70.2%) and the TCGA (60.1%) projects. When considering the mechanisms controlling allelic expression, 27.7% and 11.8% of tumors showed imbalance due to regulatory variants in cis, in the two studies respectively. Furthermore, preferential expression of the mutant allele due to cis-regulatory variation is associated with poor prognosis in the METABRIC tumors (P = 0.031). Interestingly, ER-, PR-, and HER2+ tumors showed significant preferential expression of the mutated allele in both datasets. Our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying patients of poorer prognosis, and those with low expression of the mutated allele, who will unlikely benefit from PI3K inhibitors. Furthermore, our work proposes a model of differential regulation of a critical cancer-promoting gene in breast cancer.
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