3d-multicolor-dna-fish-tool-to-study-nuclear-architecture-human

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

a-versatile-pipeline-for-analyzing-dynamic-changes-nuclear-bodies

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", INGM

Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM)

The nucleophosmin 1 gene () is the most frequently mutated gene in acute myeloid leukemia. Notably,  mutations are always accompanied by additional mutations such as those in cohesin genes , , , and  but not in the cohesin regulator, nipped B-like (). In this work, we analyzed a cohort of adult patients with acute myeloid leukemia and  mutation and observed a specific reduction in the expression of  but not in other cohesin genes. In our zebrafish model, overexpression of the mutated form of  also induced downregulation of , the zebrafish ortholog of human  To investigate the hematopoietic phenotype and the interaction between mutated  and , we generated a zebrafish model with  downregulation which showed an increased number of myeloid progenitors. This phenotype was due to hyper-activation of the canonical Wnt pathway: myeloid cells blocked in an undifferentiated state could be rescued when the Wnt pathway was inhibited by  mRNA injection or indomethacin administration. Our results reveal, for the first time, a role for  during zebrafish hematopoiesis and suggest that an interplay between  may regulate myeloid differentiation in zebrafish and humans through the canonical Wnt pathway and that dysregulation of these interactions may drive leukemic transformation.

: a new player in myeloid cell differentiation.

Despite increasing insights in genome structure organization, the role of DNA repetitive elements, accounting for more than two thirds of the human genome, remains elusive. Facioscapulohumeral muscular dystrophy (FSHD) is associated with deletion of D4Z4 repeat array below 11 units at 4q35.2. It is known that the deletion alters chromatin structure in , leading to gene up-regulation. Here we show a genome-wide role of 4q-D4Z4 array in modulating gene expression via 3D nuclear contacts. We have developed an integrated strategy of 4q-D4Z4-specific 4C-seq and chromatin segmentation analyses, showing that 4q-D4Z4 3D interactome and chromatin states of interacting genes are impaired in FSHD1 condition; in particular, genes that have lost the 4q-D4Z4 interaction and with a more active chromatin state are enriched for muscle atrophy transcriptional signature. Expression level of these genes is restored by the interaction with an ectopic 4q-D4Z4 array, suggesting that the repeat directly modulates the transcription of contacted targets. Of note, the up-regulation of atrophic genes is a common feature of several FSHD1 and FSHD2 patients, indicating that we have identified a core set of deregulated genes involved in FSHD pathophysiology.

4q-D4Z4 chromatin architecture regulates the transcription of muscle atrophic genes in facioscapulohumeral muscular dystrophy.

Epigenetic mechanisms govern the quality, the stability, and the responsiveness of transcriptional programs to the environment. This regulation is ensured via the concerted action of different players (transcription factors, "reader" and "writer" enzymes, histone marks, structural proteins, noncoding regulatory RNAs) that flow in the 3D organization of the genome. Indeed, nuclear architecture participates in the punctual and cell-type-specific regulation of transcription. Hence, the fine dissection of these mechanisms will allow a deeper understanding of the gene expression machinery. In this chapter, we propose a challenging imaging-based method to study the reciprocal interactions between chromatin-associated RNAs, genomic loci, and chromatin compartment with a procedure of 3D COMBO chrRNA-DNA-ImmunoFISH, specifically developed to preserve the nuclear integrity and topology of human primary T cells. We believe that our protocol will contribute to the improvement of epigenetic studies on the 3D nuclear structure of T cell subsets, possibly shedding light on the still hidden epigenetic players responsible for the great plasticity and functional diversification exerted by T cells.

3D COMBO chrRNA-DNA-ImmunoFISH.

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy.

How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4 T cell quiescence. LINE1-containing transcripts are derived from CD4 T cell-specific genes upregulated during T cell activation. In naive CD4 T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.

LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion.

NF1 microdeletion syndrome, accounting for 5-11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two "likely pathogenic" rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients' follow-up.

ArticleTotal : 2	Year
Publication title Cited by 3	2020
Publication title	2024

Article	Year
: a new player in myeloid cell differentiation. Haematologica\| PubMed ID: 30630974	2019
4q-D4Z4 chromatin architecture regulates the transcription of muscle atrophic genes in facioscapulohumeral muscular dystrophy. Genome research\| PubMed ID: 31097473	2019
3D COMBO chrRNA-DNA-ImmunoFISH. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32820410	2021
Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy. Pharmacological research\| PubMed ID: 34214631	2021
LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion. Nature genetics\| PubMed ID: 35039641	2022
Genetic/epigenetic effects in NF1 microdeletion syndrome: beyond the haploinsufficiency, looking at the contribution of not deleted genes. Human genetics\| PubMed ID: 38874808	2024

Federica Marasca

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