Periodic Reporting for period 2 - Chromatin3D (Chromatin Dynamics in Development and Disease)
Reporting period: 2017-01-01 to 2018-12-31
Higher order organization of chromatin results in chromosomes, the main organizing factors in the nucleus that occupy discrete territories. Most nuclear processes occur or at least being initiated onto the chromosomes which makes them the main organizing factors in the nucleus. Several proteins that are involved in the replication of DNA, gene transcription and the processing of RNA are found enriched in discrete focal structures. An emerging question is how these structures assemble and are maintained in the absence of membranes and moreover what are the kinetics of stable binding and/or rapid exchange of their components.
Overall objectives
The overall objectives of Chromatin3D were:
• To establish a European research platform of excellence in the field of chromatin dynamics and its impact on development and disease by integrating research from basic mechanisms to translational research applications.
• To create a Network dedicated to the training of young researchers promoting their independent careers, their own scientific goals and future employment prospects.
• To build durable links between the participating academic and non-academic organizations that will last beyond the duration of Chromatin3D.
Importance for society
Overall objectives
The overall objectives of Chromatin3D were:
• To establish a European research platform of excellence in the field of chromatin dynamics and its impact on development and disease by integrating research from basic mechanisms to translational research applications.
• To create a Network dedicated to the training of young researchers promoting their independent careers, their own scientific goals and future employment prospects.
• To build durable links between the participating academic and non-academic organizations that will last beyond the duration of Chromatin3D.
Importance for society
Chromatin3D ETN focused on a series of relatively short-term objectives that were achievable within the time course of the Network project. These objectives were centered to fifteen early stage researchers (ESRs) and were grouped into three broad thematic areas:
I. The epigenetic regulation of developmental processes in the context of chromatin.
CHRAC and ACF are ISWI chromatin remodeling enzymes that share the signature subunit ACF1. These complexes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. We explored the influence of the Drosophila CHRAC/ACF on transcription by complementary approaches, combining both gain- and loss-of-function strategies. Targeting the signature subunit ACF1 to multiple reporter genes inserted in many different genomic locations indicated a repressive function preferentially in the context of chromatin domains characterized by a low transcriptional output. We have confirmed that the accumulation of DNA damage in bone marrow-derived macrophages, due to the deletion or mutation of NER proteins (Ercc1-/-, XPA-/-, XPC-/-, CSBm/m), does not affect neither the allelic expression profile of Tnfα nor its total mRNA levels. SATB1 was originally described as a protein of nuclear matrix - a rigid scaffold structure inside of the nucleus. Combination of imaging experiments, RNAseq and RNA immunoprecipitation in wild type and in the Satb1 knockout thymocytes revealed interactions between SATB1 and lncRNAs such as Xist, Malat and Neat. We showed that localization of SATB1 in the nucleus is directly dependent on the presence of RNA. We have generated mouse erythroleukemic (MEL) cell lines expressing biotin-tagged FOG-1. We next isolated FOG-1 protein complexes from nuclear extracts from MEL cells expressing biotin-tagged FOG-1 by binding to streptavidin beads followed by mass spectrometry. This analysis identified known FOG-1 protein partners, such as GATA1 and the NuRD complex, as well as novel interacting partners such as CTCF and cohesins.
II. The deregulated chromatin dynamics and the cause of disease.
Proto-oncogenes of the MYC family, including MYC, MYCN and MYCL, encode related transcription factors (hereby MYC, N-MYC and L-MYC) that share an N-terminal transactivation domain and a C-terminal basic-helix-loop-helix leucine-zipper (bHLH-LZ) motif. These factors heterodimerize with a common bHLH-LZ protein, MAX, allowing sequence-specific DNA binding, with a preference toward the E-box element CACGTG, or variants thereof. Enhancers are cis-regulatory elements that play an essential role in development and can lead to disease when disrupted. However, knowledge about their function in the human genome as well as the role of genetic variation found across human populations in these regulatory regions is very limited. Several Genome Wide Association Studies (GWAS) for Central Cornea Thickness (CCT), a trait implicated in Brittle Cornea Syndrome (BCS) and a risk factor for glaucoma, have pinpointed a gene desert located on chromosome 16, upstream of the BCS gene ZNF469. Several LncRNAs found by a ncRNA microarray to have increased expression in immortalized lymphocytes derived from Sotos patients with NDS1-inactivating mutations were validated by qRT-PCR in Sotos cells. Three of these lncRNAs, including MIAT, were also found upregulated in neuroblastoma cell line LAN-I that harbours NSD1 promoter CpG island hypermethylation and transcriptional downregulation compared to neuroblastoma cell lines that do not harbour NSD1 hypermethylation. In line with the pivotal role of rhythmic LAD-circadian gene interactions in achieving circadian transcriptional attenuation, TGFbeta also strongly interfered with the serum shock-mediated synchronization of circadian transcription at several clock-controlled chromatin hubs in hEBs.
III. The development of novel approaches for the study of chromatin dynamics.
MS analysis of immunoprecipitated protein samples obtained with iDeal Chip-seq kit for TF combined with PreOmics iST kit reveals presence of the protein of interest and other interacting proteins. This suggests that combining iDeal ChIP-seq kit for TF with MS can be exploited in different research domains for identification of chromatin associated proteins and other interacting protein partners. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA-seq of macrophages stimulated with resolvin D2 (RvD2) showed similarities to transcriptional changes found during the temporal Ly6Chi to Ly6Clo macrophage transition.
I. The epigenetic regulation of developmental processes in the context of chromatin.
CHRAC and ACF are ISWI chromatin remodeling enzymes that share the signature subunit ACF1. These complexes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. We explored the influence of the Drosophila CHRAC/ACF on transcription by complementary approaches, combining both gain- and loss-of-function strategies. Targeting the signature subunit ACF1 to multiple reporter genes inserted in many different genomic locations indicated a repressive function preferentially in the context of chromatin domains characterized by a low transcriptional output. We have confirmed that the accumulation of DNA damage in bone marrow-derived macrophages, due to the deletion or mutation of NER proteins (Ercc1-/-, XPA-/-, XPC-/-, CSBm/m), does not affect neither the allelic expression profile of Tnfα nor its total mRNA levels. SATB1 was originally described as a protein of nuclear matrix - a rigid scaffold structure inside of the nucleus. Combination of imaging experiments, RNAseq and RNA immunoprecipitation in wild type and in the Satb1 knockout thymocytes revealed interactions between SATB1 and lncRNAs such as Xist, Malat and Neat. We showed that localization of SATB1 in the nucleus is directly dependent on the presence of RNA. We have generated mouse erythroleukemic (MEL) cell lines expressing biotin-tagged FOG-1. We next isolated FOG-1 protein complexes from nuclear extracts from MEL cells expressing biotin-tagged FOG-1 by binding to streptavidin beads followed by mass spectrometry. This analysis identified known FOG-1 protein partners, such as GATA1 and the NuRD complex, as well as novel interacting partners such as CTCF and cohesins.
II. The deregulated chromatin dynamics and the cause of disease.
Proto-oncogenes of the MYC family, including MYC, MYCN and MYCL, encode related transcription factors (hereby MYC, N-MYC and L-MYC) that share an N-terminal transactivation domain and a C-terminal basic-helix-loop-helix leucine-zipper (bHLH-LZ) motif. These factors heterodimerize with a common bHLH-LZ protein, MAX, allowing sequence-specific DNA binding, with a preference toward the E-box element CACGTG, or variants thereof. Enhancers are cis-regulatory elements that play an essential role in development and can lead to disease when disrupted. However, knowledge about their function in the human genome as well as the role of genetic variation found across human populations in these regulatory regions is very limited. Several Genome Wide Association Studies (GWAS) for Central Cornea Thickness (CCT), a trait implicated in Brittle Cornea Syndrome (BCS) and a risk factor for glaucoma, have pinpointed a gene desert located on chromosome 16, upstream of the BCS gene ZNF469. Several LncRNAs found by a ncRNA microarray to have increased expression in immortalized lymphocytes derived from Sotos patients with NDS1-inactivating mutations were validated by qRT-PCR in Sotos cells. Three of these lncRNAs, including MIAT, were also found upregulated in neuroblastoma cell line LAN-I that harbours NSD1 promoter CpG island hypermethylation and transcriptional downregulation compared to neuroblastoma cell lines that do not harbour NSD1 hypermethylation. In line with the pivotal role of rhythmic LAD-circadian gene interactions in achieving circadian transcriptional attenuation, TGFbeta also strongly interfered with the serum shock-mediated synchronization of circadian transcription at several clock-controlled chromatin hubs in hEBs.
III. The development of novel approaches for the study of chromatin dynamics.
MS analysis of immunoprecipitated protein samples obtained with iDeal Chip-seq kit for TF combined with PreOmics iST kit reveals presence of the protein of interest and other interacting proteins. This suggests that combining iDeal ChIP-seq kit for TF with MS can be exploited in different research domains for identification of chromatin associated proteins and other interacting protein partners. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA-seq of macrophages stimulated with resolvin D2 (RvD2) showed similarities to transcriptional changes found during the temporal Ly6Chi to Ly6Clo macrophage transition.
The Chromatin3D Network brought together European institutions, the industry and students. This unique combination of talents increased the overall quality of academic and industrial training of young European researchers, which is being formed in Europe and will likely continue to deliver their knowledge within Europe in the future. The long-term implications on the fellows’ career prospects deemed significant: The training program enabled ESRs to develop and further extend a solid scientific basis, critical judgement and discipline in their research activities allowing them to reach scientific maturity and independence. The combination of basic biology and cutting-edge technologies of the training programme ensured that the fellows graduated as a new breed of scientists trained in facing the challenges of contemporary post-genomic biology using advanced technological tools that are widely applicable in both academia and industry, thereby substantially enhancing their research career prospects.