Monica Beltrame

The double life of HMGB1 chromatin protein: architectural factor and extracellular signal

The High Mobility Group Box (HMGB) chromosomal proteins have been known and studied for a long time, but we have only recently started to understand their biological functions. They now have a clear reputation for being important architectural factors: they facilitate the assembly of site‐specific DNA binding proteins to their cognate binding sites within chromatin. Beyond this intranuclear function, they also have an extracellular function, which will be the prime focus of this short review. ### The HMGB family: structure, expression and nuclear function The HMGB family comprises the three proteins HMGB1 (previously HMG1), HMGB2 (previously HMG2) and HMGB3 (previously HMG4 or HMG2b) (Bustin, 2001). The structure of these three proteins is highly conserved (>80% amino acid identity), and their biochemical properties are so far indistinguishable. HMGBs are composed of three different domains. The two homologous DNA binding domains, HMG boxes A and B, are each ∼75 amino acids in length. The C‐terminal domain is highly negatively charged, consisting of a continuous stretch of glutamate or aspartate residues, and is longest in HMGB1 and shortest in HMGB3 (reviewed in Bustin, 1999; Bianchi and Beltrame, 2000). HMGB1 is ubiquitous and only 10 times less abundant than core histones, at ∼106 molecules per typical mammalian cell. Expression of the other two family members is more restricted: HMGB3 is only expressed to a significant amount during embryogenesis (Vaccari et al ., 1998); HMGB2 is widely expressed during embryonic development, but restricted mainly to lymphoid organs and testis in the adult mouse (Ronfani et al ., 2001). The localization of these proteins in most cells is nuclear. In their nuclear identity, HMGB1 and HMGB2 bind to the minor groove of DNA, causing a local distortion of the double helix. They have little or no sequence preference, and they are recruited to the site of action by specific DNA binding proteins. HMGB1 has …

Protein HU binds specifically to kinked DNA

We have purified the main four‐way junction DNA‐binding protein of Escherichia coli, and have found It to be the well‐known HU protein. HU protein recognizes with high‐affinity one of the angles present in the junction, a molecule with the shape of an X. Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound. HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate. All these properties are analogous to the properties of the mammalian chromatin protein HMG1. We suggest that HU is a prokaryotic HMG1‐like protein rather than a histone‐like protein.

Upwardly mobile proteins: Workshop: The role of HMG proteins in chromatin structure, gene expression and neoplasia

High Mobility Group proteins (HMGs) are a set of chromatin proteins first identified in the 1970s because they are very abundant and run fast on SDS–PAGE. In these and other properties they resemble histones. And like histones, which have seen a resurgence of interest thanks to the discovery that their modification modulates transcription, HMGs are staging a comeback. They now appear to be important and versatile players in the same complex plot: they regulate the expression of genes in normal or pathological conditions. About a hundred researchers from four continents gathered for 2 days (May 1 and 2, 2000) at the Lister Hill Center of the NIH, Bethesda, to discuss HMGs. Here we report on both the general picture and some of the most novel results (at least to us). Only work published in the last year is cited in the References; for background information, excellent starting points are the reviews by Bustin (1999) and Wegner (1999). ### A rose is a rose is a rose (but there are 2 × 3 HMGs) HMGs were discovered by the British scientist H.M. Goodwin, which has led some to speculate that their name reflects the initials of the discoverer, or of Her Majestys Government. Today, the name refers to two classes of proteins: the canonical HMGs, and HMG‐motif proteins. Canonical HMGs are ubiquitous to eukaryotes but are absent in eubacteria and archaea. They can be divided into three groups that are completely dissimilar from one another at the level of sequence and structure, but are internally homogeneous: the HMG1/2, HMG‐14/17, and HMG‐I/Y families (Table 1). Each family is characterized by a functional sequence motif: the HMG box, the nucleosome binding domain, or the AT‐hook (Figure 1). HMG‐motif proteins contain one of these functional motifs, but the rest of the sequence is different. Figure 1. Structure of AT‐hooks and HMG boxes, as determined by the laboratory of M. Clore (Bethesda, …

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

ABSTRACT V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

Flexing DNA: HMG-Box Proteins and Their Partners

The initiation of transcription by RNA polymerase II is controlled by the assembly of the basal transcription machinery, which integrates inputs from transcription factors bound to promoters and enhancers. Overall, a typical gene may depend on the binding of tens of transcription factors to its controlling-sequence elements. Combinatorial assembly ensures that each gene can exhibit a distinctive and sometimes variable pattern of expression, despite the fact that the number of transcription factors is much lower than the number of genes.

SoxF genes: Key players in the development of the cardio-vascular system

SoxF genes (Sox7, Sox17 and Sox18) encode a group of transcription factors that have a pivotal role in cardio-vascular development. SOXF factors orchestrate endothelial cell fate or direct cell differentiation in developing heart, blood vessels and lymphatic vessels. Their roles are highly conserved throughout animal evolution. SOXF activity is finely tuned with a variety of cell type-specific co-factors and partner proteins to effect transcription of genes critical for endothelial cell phenotype and function. Because SOXF factors play a central role in cardiogenesis, vasculogenesis and lymphangiogenesis, SOXF gene mutations figure prominently in the aetiology of human vascular disease.

Aβ peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis

Cerebral amyloid angiopathy (CAA) caused by amyloid β (Aß) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Aß properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Aß peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild‐type (WT) Aß and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC50 of 6.1 and 4.7 μM, respectively. The 2.5 μM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. ß‐Galactosidase staining and the cyclindependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC50 of 12.3 and 8.8 μM, respectively. The 5 μM concentration, devoid of acute effects on the endothelium, was applied chronically to long‐term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with ß‐galactosidase accumulation, down‐regulation of telomerase reversetranscriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Aß treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Aß‐induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.—Donnini, S., Solito, R., Cetti, E., Corti, F., Giachetti, A., Carra, S., Beltrame, M., Cotelli, F., Ziche, M. Aß peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEBJ. 24, 2385–2395 (2010). www.fasebj.org

Expression patterns of zebrafish sox11A, sox11B and sox21.

We have cloned three sox genes in zebrafish (Danio rerio), one related to human and chicken SOX21, and two related to mammalian and chicken Sox-11. Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation and become restricted later to the developing central nervous system (CNS); expression in adults is undetectable. sox21 is expressed in the forebrain, midbrain and hindbrain, but maximally at the midbrain-hindbrain junction; sox11A,B have a widespread and dynamic expression in the CNS, but in contrast to sox21 are absent at the midbrain-hindbrain boundary.

Engineering of a light-gated potassium channel

An optogenetic tool to silence neurons Potassium channels in the cell membrane open and close in response to molecular signals to alter the local membrane potential. Cosentino et al. linked a light-responsive module to the pore of a potassium channel to build a genetically encoded channel called BLINK1 that is closed in the dark and opens in response to low doses of blue light. Zebrafish embryos expressing BLINK1 in their neurons changed their behavior in response to blue light. Science, this issue p. 707 Blue light opens a channel to silence excitable neurons. The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K+) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light–induced K+ channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K+ channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K+ selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K+ equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.

Ectopic expression and knockdown of a zebrafish sox21 reveal its role as a transcriptional repressor in early development.

Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the oocyte and is mainly expressed at the developing midbrain-hindbrain boundary from the onset of neurulation. In order to understand its role in vivo, we ectopically expressed sox21a by microinjection. Ectopic expression of full length sox21a leads to dorsalization of the embryos. A subset of the dorsalized embryos shows a partial axis splitting, and hence an ectopic neural tube, as an additional phenotype. At gastrulation, injected embryos show expansion of the expression domains of organizer-specific genes, such as chordin and goosecoid. Molecular markers used in somitogenesis highlight that sox21a-injected embryos have shortened AP axis, undulating axial structures, enlarged or even radialized paraxial territory. The developmental abnormalities caused by ectopic expression of sox21a are suggestive of defects in convergence-extension morphogenetic movements. Antisense morpholino oligonucleotides, designed to functionally knockdown sox21a, cause ventralization of the embryos. Moreover, gain-of-function experiments with chimeric constructs, where Sox21a DNA-binding domain is fused to a transcriptional activator (VP16) or repressor (EnR) domain, suggests that zebrafish Sox21a acts as a repressor in dorso-ventral patterning.