Claudio Brancolini

University of Udine | UNIUD · Department of Medical and Biological Sciences

Controlling access to genomic information and maintaining its stability are key aspects of cell life. Histone acetylation is a reversible epigenetic modification that allows access to DNA and the assembly of protein complexes that regulate mainly transcription but also other activities. Enzymes known as histone deacetylases (HDACs) are involved in...

Cinnamic acids are an important class of phenolic compounds, which have many beneficial effects on human health but are also interesting synthetic intermediates thanks to the presence of several reactive sites. While studying the reactivity of cinnamic acids with diazonium salts from aromatic amines, an unexpected reactivity has been discovered, le...

Super-enhancers evolve as elements at the top of the hierarchical control of gene expression. They are important end-gatherers of signaling pathways that control stemness, differentiation or adaptive responses. Many epigenetic regulations focus on these regions, and not surprisingly, during the process of tumorigenesis, various alterations can acco...

Epigenetic mechanisms have a main impact on cancer regulation and on microenvironment control. In the context of leiomyosarcoma (LMS), a highly aggressive and immunologically cold cancer, class IIa histone deacetylases (HDACs) and myocyte enhancer factor (MEF2) form a repressive complex that leads to the reorganization of epigenetic landscape. We h...

Leiomyosarcoma (LMS) is aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We foun...

Cancer cells must adapt to the hostile conditions of the microenvironment in terms of nutrition, space, and immune system attack. Mutations of DNA are the drivers of the tumorigenic process, but mutations must be able to hijack cellular functions to sustain the spread of mutant genomes. Transcriptional control is a key function in this context and...

Non-muscle-invasive bladder cancer (NMIBC) is the most frequently diagnosed bladder cancer and intravesical instillation of Bacillus Calmette-Guérin (BCG) plus transurethral resection (TUR) is the standard therapeutic approach [1]. Since delayed interventions are often associated with lower patient survival, knowing which tumors develop aggressive...

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a dockin...

Background: Histone deacetylase 4 (HDAC4) is a stress-responsive factor that mediates multiple cellular responses. As a member of class IIa HDACs, HDAC4 shuttles between the nucleus and the cytoplasm; however, HDAC4 cytoplasmic functions have never been fully investigated. Duchenne muscular dystrophy (DMD) is a genetic, progressive, incurable diso...

The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of muscle cells, neurons and hematopoietic cells. By functioning in physiological feedback loops, Mef2 TFs promote the transcription of their repressor, Hdac9, thereby providing temporal control of Mef2-driven differentiation. Disruption of this feedback is...

Background Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not comp...

Background: In the breast, the pleiotropic epigenetic regulator HDAC7 can influence stemness. Materials & Methods: The authors used MCF10 cells knocked-out for HDAC7 to explore the contribution of HDAC7 to IGF1 signaling. Results: HDAC7 buffers H3K27ac levels at the IGFBP6 and IGFBP7 genomic loci and influences their expression. In this manner, HDA...

p>Leiomyosarcomas (LMS) are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of LMS recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low re...

Simple Summary In this review article, we will deepen the topic of cancer cell communication with the surrounding non-cancerous cells. In particular, the non-mutational events that modified gene expression, namely “epigenetics”, involved in cell–cell interaction will be the center of this work. Many studies have described the mechanism of back-and-...

Understanding how an epigenetic regulator drives different cellular responses can be a tricky task. Very often, their activities are modulated by large multiprotein complexes, the composition of which is context- and time-dependent. As a consequence, experiments aimed to unveil the functions of an epigenetic regulator can provide different outcomes...

To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive resp...

To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact on normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive r...

The epigenome of senescent cells is characterized by a deep redistribution of H3K27 acetylation. H3K27 is target of class IIa Histone Deacetylases (HDAC4, 5, 7, 9) as part of large repressive complexes. We report here that, among class IIa HDACs, HDAC4 is post-transcriptionally downregulated during senescence and aging. HDAC4 knock-out (KO) trigger...

Senescence is the end point of a complex cellular response that proceeds through a set of highly regulated steps. Initially, the permanent cell-cycle arrest that characterizes senescence is a pro-survival response to irreparable DNA damage. The maintenance of this prolonged condition requires the adaptation of the cells to an unfavorable, demanding...

Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 t...

Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation...

HDAC7 is a pleiotropic transcriptional co‐regulator that controls different cellular fates. Here, we demonstrate that in human mammary epithelial cells, HDAC7 sustains cell proliferation and favors a population of stem‐like cells, by maintaining a proficient microenvironment. In particular, HDAC7 represses a repertoire of cytokines and other enviro...

Fig. S1. HDAC7‐S/A triggers senescence similarly to HDAC4‐TM.

Expression of the class IIa HDACs is frequently altered in different human cancers. In mouse models these transcriptional repressors can trigger transformation, acting as bona fide oncogenes. Whether class IIa HDACs also exhibit transforming activities in human cells is currently unknown. We infected primary human fibroblasts with retroviruses to i...

While MEF2 transcription factors are well known to cooperate in orchestrating cell fate and adaptive responses during development and adult life, additional studies over the last decade have identified a wide spectrum of genetic alterations of MEF2 in different cancers. The consequences of these alterations, including triggering and maintaining the...

Diaryldienone derivatives with accessible β-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmar...

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, s...

The 85 common MEF2-target genes. The list of the genes significantly regulated by MEF2A e MEF2D silencing in both SK-LMS-1 and SK-UT-1 cells. Values are indicated as mean fold change relative to the control. A prediction of the binding of MEF2 TFs on chromatin was done by scrutinizing all published BED files of ChIP-seq data: ENCFF148PLM, ENCFF001T...

Characteristics of the selected atypical and classical MEF2-target genes. (XLSX)

MEF2D-HDAC9 complex. The MEF2D-HDAC9 complexes were immunoprecipitated from the different cell lines using 1μg of anti-MEF2D, or anti-FLAG antibodies, as a control. Immunocomplexes were subjected to immunoblotting using the anti-MEF2D and HDAC9 antibodies. (TIF)

Immunohistochemistry analysis. (XLSX)

Roles of MEF2A and of MEF2D in tumor cells invasion. Fluorescence analysis of Matrigel invading SK-LMS-1 and SK-UT-1 cells expressing the indicated shRNAs and stained with Hoechst 33342. Bar = 100μM. (TIF)

Silencing of MEF2D isoforms in SK-LMS-1. A) qRT-PCR analysis of the mRNAs expression levels of two alternative isoforms of MEF2D (α1 and α2) in SK-LMS-1 cells expressing the indicated isoform-specific shRNAs. mRNA levels are relative to control shRNA. Data are presented as mean ± SD; n = 3. B) Immunoblot analysis of the MEF2D isoforms levels in SK-...

MEF2D and HDAC4 co-occupancy. 6x106 cells were employed. First immunoprecipitations were conducted ON with 2μg of anti-MEF2D or anti-FLAG antibodies. Protein-DNA complexes were collected with 8μl of protein A magnetic beads (ZymoMag, Zymo research) and washed twice with RIPA and TE. Beads were incubated for 30’ at 37°C in Re-Chip elution buffer (1×...

Characterization of the new MEF2-target genes. A) qRT-PCR analysis of the mRNA expression levels of the identified atypical and classical MEF2-target genes in SK-LMS-1 cells expressing the shRNAs against MEF2D. mRNA levels are relative to control shRNA. GAPDH was used as control. Data are presented as mean ± SD; n = 3. B) qRT-PCR analysis of the mR...

Role of MEF2 in controlling histone H3K27 acetylation. A) Chromatin was immunoprecipitated from SK-LMS-1 or SK-UT-1 cells WT or KD for MEF2D, using the anti-H3K27ac antibody. Normal rabbit IgGs were used as control. The MEF2 binding site (arrowheads), the amplified region and the TSS (arrows) are indicated for each tested atypical gene. Data are pr...

Role of MEF2 in controlling histone H3K4 methylation. A) Chromatin was immunoprecipitated from SK-LMS-1 or SK-UT-1 cells WT or KD for MEF2D, using the anti-H3K4me3 antibody. Normal rabbit IgGs were used as control. The MEF2 binding site (arrowheads), the amplified region and the TSS (arrows) are indicated for each tested atypical gene. Data are pre...

CRISPR/Cas9 mediated KO of HDAC4 and HDAC9. A) Schematic representation of HDAC9 genomic organization with indicated: the exons (vertical bars), the introns (junctions between the bars) and the PAM sequences utilized for the CRISPR approach. B) Genomic sequences of the HDAC9-/- SK-UT-1 cells used in this study. The sequence of HDAC9 genomic region...

Genes comprised in the MEF2 signature. (XLSX)

MEF2A silencing causes opposite effects in SK-LMS-1 and SK-UT-1 cells. A) MEF2A expression was silenced by lentiviral infection using two different shRNA (#4 and #5). Immunoblot analysis of MEF2D and CDKN1A levels in SK-LMS-1 cells expressing the control shRNA or two different shRNAs against MEF2A. Actin was used as loading control. B) qRT-PCR anal...

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T-cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2-c][1,3]benzothiazin-6-imine scaffold was studied that is completely different from the traditional concept of HDAC...

Metabolic adaptations are emerging as common traits of cancer cells and tumor progression. In vitro transformation of NIH 3T3 cells allows the analysis of the metabolic changes triggered by a single oncogene. In this work, we have compared the metabolic changes induced by H-RAS and by the nuclear resident mutant of histone deacetylase 4 (HDAC4). RA...

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors tr...

In response to environmental cues, enzymes that influence the functions of proteins, through reversible post-translational modifications supervise the coordination of cell behavior like orchestral conductors. Class IIa histone deacetylases (HDACs) belong to this category. Even though in vertebrates these deacetylases have discarded the core enzymat...

The MEF2-HDAC axis is a master regulator of different developmental programs and adaptive responses in adults. In this manuscript we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is up-reg...

MEF2s are pleiotropic transcription factors (TFs), which supervise multiple cellular activities. During the cell-cycle MEF2s are activated at the G0/G1 transition to orchestrate the expression of the immediate early genes in response to growth factors stimulation. Here we show that, in human and murine fibroblasts, MEF2 activities are down-regulate...

Bis arylidenecycloalkanones structurally related to the Non Selective Isopeptidase Inhibitor G5 were synthesized and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted arylidene groups confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydro...

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis...

The prospect of intervening, through the use of a specific molecule, with a cellular alteration responsible for a disease, is a fundamental ambition of biomedical science. Epigenetic-based therapies appear as a remarkable opportunity to impact on several disorders, including cancer. Many efforts have been made to develop small molecules acting as i...

Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 an...

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising molecule for anti-cancer therapies. Unfortunately, cancer cells frequently acquire resistance to rhTRAIL. Various co-treatments have been proposed to overcome apoptosis resistance to TRAIL. Here we show that downregulation of the deISGylase USP18 sensitizes cancer...

The MEF2-class IIa histone deacetylase (HDAC) axis operates in several differentiation pathways and in numerous adaptive responses. We show here that nuclear active HDAC4 and HDAC7 display transforming capability. HDAC4 oncogenic potential depends on the repression of a limited set of genes, most of which are MEF2 targets. Genes verified as targets...

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regula...

MEF2s transcription factors and class IIa HDACs compose a fundamental axis for several differentiation pathways. Functional relationships between this axis and cancer are largely unexplored. We have found that class IIa HDACs are heterogeneously expressed and display redundant activities in breast cancer cells. Applying gene set enrichment analysis...

Type 1 diabetes (T1D) is an autoimmune disease targeting pancreatic beta cells. Genome-wide association studies and gene expression analysis identified interferon (IFN)-driven gene networks as crucial pathways in the pathogenesis of T1D. IFNs are linked to the response to viral infections and might contribute to the initiation of the autoimmune pro...

IFNs are cytokines that segregate viral infections, modulate the immune responses and influence tumor cells survival. These options are under the control of ISGs (Interferon Stimulated Genes) which expression is propelled by IFNs. To the ISGs family belong all the components of the molecular machinery that modifies proteins by the addition of the u...

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring au...

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring au...

Deletion of type I IFN genes and resistance to apoptosis induced by type I IFNs are common in glioblastoma. Here we have investigated the importance of the constitutive weak IFN-signaling in the apoptotic response to IFN-α in glioblastoma cells. U87MG cells hold a deletion of type I IFN genes, whereas in T98G cells the spontaneous IFN signaling is...

Caspases are critical regulators of the apoptotic program, responsible for the harmonic dismantling of the cell. Cell death can occur by way of different options (necroptosis, necrosis, extreme autophagy) but once caspases are fully engaged it will take the apoptotic route. Hence, in general, caspase activity is inversely proportional to cell viabi...