Hmga2 protein loss alters nuclear envelope and 3D chromatin structure.

BACKGROUND: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. RESULTS: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. CONCLUSIONS: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.

Activation of Non-Canonical Autophagic Pathway through Inhibition of Non-Integrin Laminin Receptor in Neuronal Cells.

To fight neurodegenerative diseases, several therapeutic strategies have been proposed that, to date, are either ineffective or at the early preclinical stages. Intracellular protein aggregates represent the cause of about 70% of neurodegenerative disorders, such as Alzheimer's disease. Thus, autophagy, i.e., lysosomal degradation of macromolecules, could be employed in this context as a therapeutic strategy. Searching for a compound that stimulates this process led us to the identification of a 37/67kDa laminin receptor inhibitor, NSC48478. We have analysed the effects of this small molecule on the autophagic process in mouse neuronal cells and found that NSC48478 induces the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) into the LC3-phosphatidylethanolamine conjugate (LC3-II). Interestingly, upon NSC48478 treatment, the contribution of membranes to the autophagic process derived mainly from the non-canonical m-TOR-independent endocytic pathway, involving the Rab proteins that control endocytosis and vesicle recycling. Finally, qRT-PCR analysis suggests that, while the expression of key genes linked to canonical autophagy was unchanged, the main genes related to the positive regulation of endocytosis (pinocytosis and receptor mediated), along with genes regulating vesicle fusion and autolysosomal maturation, were upregulated under NSC48478 conditions. These results strongly suggest that 37/67 kDa inhibitor could be a useful tool for future studies in pathological conditions.

MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development.

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial-mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression.

Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity.

HMGA Proteins in Stemness and Differentiation of Embryonic and Adult Stem Cells.

HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available experimental data on HMGA1 and HMGA2 functions in governing embryonic and adult stem cell fate. Moreover, based on the available evidence, we will aim to outline how HMGA expression is regulated in different contexts and how these two proteins contribute to the regulation of gene expression and chromatin architecture in stem cells.