Two major types of leukemogenic BCR-ABL fusion proteins are p190and p210. Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. We took an integrated approach by coupling protein-protein interaction analysis using biotinylation identification with global phosphorylation analysis to investigate the differences in signaling between these two fusion proteins. Our findings suggest that p190 and p210 differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210, we observed an increased engagement of molecules active proximal to the membrane and in the case of p190, an engagement of molecules of the cytoskeleton. These differences in signaling could underlie the distinct leukemogenic process induced by these two protein variants.

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

Summary Non-random, dynamic three-dimensional organization of the nucleus is important for regulation of gene expression. Numerous studies using chromosome conformation capture strategies have uncovered ensemble organizational principles of individual chromosomes, including organization into active (A) and inactive (B) compartments. In addition, large inactive regions of the genome appear to be associated with the nuclear lamina, the so-called Lamina Associated Domains (LADs). However, the interrelationship between overall chromosome conformation and association of domains with the nuclear lamina remains unclear. In particular, the 3D organization of LADs within the context of the entire chromosome has not been investigated. In this study, we describe “chromosome conformation paints” to determine the relationship in situ between LAD and non-LAD regions of the genome in single cells. We find that LADs organize into constrained and compact regions at the nuclear lamina, and these findings are supported by an integrated analysis of both DamID and Hi-C data. Using a refined algorithm to identify active (A) and inactive (B) compartments from Hi-C data, we demonstrate that the LADs correspond to the B compartment. We demonstrate that in situ single cell chromosome organization is strikingly predicted by integrating both Hi-C and DamID data into a chromosome conformation model. In addition, using the chromosome conformation paints, we demonstrate that LAD (and B-compartment) organization is dependent upon both chromatin state and Lamin A/C. Finally, we demonstrate that small regions within LADs escape the repressive regime at the peripheral zone to interact with the A-compartment and are enriched for both transcription start sites (TSSs) and active enhancers.

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Objectives Although uterine cancer is the fourth most common cause for cancer death in women worldwide, the molecular underpinnings of tumor progression remain poorly understood. The High Mobility Group A1 (HMGA1) gene is overexpressed in aggressive cancers and high levels portend adverse outcomes in diverse tumors. We previously reported that Hmga1 transgenic mice develop uterine tumors with complete penetrance. Because HMGA1 drives tumor progression by inducing matrix metalloproteinase (MMP) and other genes involved in invasion, we explored the HMGA1-MMP-2 pathway in uterine cancer. Methods To investigate MMP-2 in uterine tumors driven by HMGA1, we used a genetic approach with mouse models. Next, we assessed HMGA1 and MMP-2 expression in primary human uterine tumors, including low-grade carcinomas (endometrial endometrioid) and more aggressive tumors (endometrial serous carcinomas, uterine carcinosarcomas/malignant mesodermal mixed tumors). Results Here, we report for the first time that uterine tumor growth is impaired in Hmga1a transgenic mice crossed on to an Mmp-2 deficient background. In human tumors, we discovered that HMGA1 is highest in aggressive carcinosarcomas and serous carcinomas, with lower levels in the more indolent endometrioid carcinomas. Moreover, HMGA1 and MMP-2 were positively correlated, but only in a subset of carcinosarcomas. HMGA1 also occupies the MMP-2 promoter in human carcinosarcoma cells. Conclusions Together, our studies define a novel HMGA1-MMP-2 pathway involved in a subset of human carcinosarcomas and tumor progression in murine models. Our work also suggests that targeting HMGA1 could be effective adjuvant therapy for more aggressive uterine cancers and provides compelling data for further preclinical studies.

Myeloproliferative neoplasms (MPN) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the High Mobility Group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is up-regulated in MPN with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and bone marrow. RNA- and chromatin immunoprecipitation-sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and promising therapeutic target to treat or prevent disease progression.

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.