Regulation of XMyoDa transcription by the binding of XMEF2A to the TATA box

MEF2 is a $\underline{\rm m}$yocyte-specific $\underline{\rm e}$nhancer-binding $\underline{\rm f}$actor that binds a conserved DNA sequence, CTA(A/T)$\sb4$TAG. A MEF2 binding site in the XMyoDa promoter overlaps with the TATA box and is required for muscle specific expression. To examine the potential role of MEF2 in the regulation of MyoD transcription during early development, the appearance of MEF2 binding activity in developing Xenopus embryos was analyzed with the electrophoretic mobility shift assay. Two genes were isolated from a X. Laevis stage 24 cDNA library that encode factors that bind the XMyoDa TFIID/MEF2 site. Both genes are highly homologous to each other, belong to the MADS ($\underline{\rm M}$CM1-$\underline{\rm A}$rg80-agamous-$\underline{\rm d}$eficiens-$\underline{\rm S}$RF) protein family, and most highly related to the mammalian MEF2A gene, hence they are designated as XMEF2A1 and XMEF2A2. Proteins encoded by both cDNAs form specific complexes with the MEF2 binding site and show the same binding specificity as the endogenous MEF2 binding activity. XMEF2A transcripts accumulate preferentially in developing somites after the appearance of XMyoD transcripts. XMEF2 protein begins to accumulate in somites at tailbud stages. Transcriptional activation of XMyoD promoter by XMEF2A required only the MADS box and MEF2-specific domain when XMEF2A is bound at the TATA box. However, a different downstream transactivation domain was required when XMEF2A activates transcription through binding to multiple upstream sites. These results suggest that different activation mechanisms are involved, depending on where the factor is bound. Mutations in several basic amino acid clusters in the MADS box inhibit DNA binding suggesting these amino acids are essential for DNA binding. Mutation of Thr-20 and Ser-36 to the negatively charged amino acid residue, aspartic acid, abolish DNA binding. XMEF2A activity may be regulated by phosphorylation of these amino acids. A dominant negative mutant was made by mutating one of the basic amino acid clusters and deleting the downstream transactivation domain. In vivo roles of MEF2 in the regulation of MyoD transcription were investigated by overexpression of wild type MEF2 and dominant negative mutant of XMEF2A in animal caps and assaying for the effects on the level of expression of MyoD genes. Overexpression of MEF2 activates the transcription of endogenous MyoD gene family while expression of a dominant negative mutant reduces the level of transcription of XMRF4 and myogenin genes. These results suggest that MEF2 is downstream of MyoD and Myf5 and that MEF2 is involved in maintaining and amplifying expression of MyoD and Myf5. MEF2 is upstream of MRF4 and myogenin and plays a role in activating their expression. ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^

Human placental lactogen enhancer: Tissue specificity and binding with specific proteins

Human placental lactogen (hPL) is a 22,000 dalton protein hormone produced in the placenta. The physiological actions of hPL are not well understood but its major activity is to regulate both maternal and fetal metabolism. hPL stimulates maternal lipolysis increasing free fatty acids in the maternal blood, allowing their use as an energy source by the mother, and sparing glucose for the fetus. It may also act as a growth promoting hormone for the fetus. hPL is produced in increasing amounts as pregnancy progresses. At term, hPL accounts for 10% of protein and 5% of total RNA in the placenta. This high level of hPL production is tissue-specific, as hPL is only produced in the placenta by syncytiotrophoblast cells.^ The objective of this work was to understand the mechanism by which such high levels of hPL are produced in a tissue-specific manner. A transcriptional enhancer found 2.2 kb 3$\sp\prime$ to one of the hPL genes (hPL$\sb3$) may explain the regulation of hPL expression. Transient transfection experiments using the hPL-producing human choriocarcinoma cell line JEG-3 localized the hPL enhancer to a 138 bp core element. This 138 bp sequence was found to be tissue specific in its actions as it did not promote transcription in heterologous cell lines. Gel mobility shift assays showed the hPL enhancer interacts specifically with nuclear proteins unique to hPL-producing cells. Within the 138 bp enhancer a 22 bp region was shown to be protected from DNase I digestion due to binding of proteins derived from placental nuclear extracts. Proteins binding this region of the enhancer may be instrumental in the tissue specific activity of the hPL enhancer. ^

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

Sp1 up-regulates cAMP-response-element-binding protein expression during retinoic acid-induced mucous differentiation of normal human bronchial epithelial cells.

CREB [CRE (cAMP-response element)-binding protein] is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA (retinoic acid) rapidly activates CREB without using RARs (RA receptors) or RXRs (retinoid X receptors) in NHTBE cells (normal human tracheobronchial epithelial cells). However, little is known about the role of RA in the physiological regulation of CREB expression in the early mucous differentiation of NHTBE cells. In the present study, we report that RA up-regulates CREB gene expression and that, using 5'-serial deletion promoter analysis and mutagenesis analyses, two Sp1 (specificity protein 1)-binding sites located at nt -217 and -150, which flank the transcription initiation site, are essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nt -119 and -98 contributed to basal promoter activity. Interestingly, RA also up-regulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using siRNA (small interfering RNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA up-regulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in up-regulating human CREB gene expression. This result implies that co-operation of these two transcription factors plays a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells.

A family of fibrinogen-binding MSCRAMMs from Enterococcus faecalis.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of beta-sheets with only a minor proportion of alpha-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1.

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.

Functional analysis of the amine substrate specificity domain of pepper tyramine and serotonin N-hydroxycinnamoyltransferases.

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.