Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

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.

Active water in protein-protein communication within the membrane: the case of SRII-HtrII signal relay.

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

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. ^

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. ^

The Draft Assembly of the Radically Organized Stylonychia lemnae Macronuclear Genome.

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

The multifunctional FUS protein: Characterization of the biological effects of its depletion

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line, we have performed a whole transcriptome analysis by RNA deep sequencing. Preliminary results show that FUS depletion affects both expression and alternative splicing levels of several RNAs. When FUS is depleted we observed 330 downregulated and 81 upregulated genes. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, to further characterize the FUS-depleted cell line we have performed growth proliferation and survival assays. From these experiments emerge that FUS-depleted cells display growth proliferation alteration. In order to explain this observation, we have tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than controls. Currently, we are looking for putative candidate targets causing this phenotype. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

The FUS protein is required for cell proliferation

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed and highly conserved RNA binding protein, has been linked to a variety of cellular processes from mRNA processing to DNA repair. However, the precise function of FUS is not well understood. Recently, mutations in the FUS gene have been identified in familial and sporadic patients of Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disorder characterized by dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS that efficiently depletes the protein. In order to characterize this cell line, we have characterized the poly(A) fraction by RNA deep sequencing. Preliminary results show that FUS depletion affects both mRNA expression and alternative splicing. Upon FUS depletion 330 genes are downregulated and 81 are upregulated. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, we are currently characterizing how FUS depletion affects cell proliferation and survival. We find that the lack of FUS impairs cell proliferation but does not induce apoptosis. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

Protein profiling of urine from dogs with renal disease using ProteinChip analysis

Measurement of total urinary proteins in individuals that tested positive by urinary dipstick is a typical method for assessing the presence of potentially serious renal disorders. In the absence of such overt proteinuria, however, measurement of specific urinary proteins may be useful in the diagnosis of nephropathies and may provide greater insight into the pathogenesis. The urine of 28 dogs (16 with renal disease and 12 healthy) was evaluated to determine whether specific low-molecular-weight proteins or the pattern of protein excretion could also be used as a marker of tubular dysfunction in dogs. Specific proteins were assessed by immunological methods, whereas protein profiles were determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MS). In particular, changes in the excretion of retinol-binding protein (RBP) and Tamm-Horsfall protein (THP) appear to be of clinical relevance in the diagnosis of canine kidney diseases. The pattern of urinary protein and peptides revealed specific changes in abundance in dogs with renal disease at molecular masses (kD) of 11.58, 12.41, 12.60, 14.58, 20.95 (RBP), 27.85, and 65.69 (albumin). In conclusion, comparable proteins as in humans might be used as urinary markers for proximal (RBP) and distal (THP) tubular dysfunction in dogs. Surface-enhanced laser desorption/ionization time-of-flight MS is a promising tool for the study of kidney physiology and pathophysiology and might aid in the discovery of new biomarkers of renal disease.

The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Specificities of Caenorhabditis elegans and human hairpin binding proteins for the first nucleotide in the histone mRNA hairpin loop.

The 3' ends of animal replication-dependent histone mRNAs are formed by endonucleolytic cleavage of the primary transcripts downstream of a highly conserved RNA hairpin. The hairpin-binding protein (HBP) binds to this RNA element and is involved in histone RNA 3' processing. A minimal RNA-binding domain (RBD) of approximately 73 amino acids that has no similarity with other known RNA-binding motifs was identified in human HBP [Wang Z-F et al., Genes & Dev, 1996, 10:3028-3040]. The primary sequence identity between human and Caenorhabditis elegans RBDs is 55% compared to 38% for the full-length proteins. We analyzed whether differences between C. elegans and human HBP and hairpins are reflected in the specificity of RNA binding. The C. elegans HBP and its RBD recognize only their cognate RNA hairpins, whereas the human HBP or RBD can bind both the mammalian and the C. elegans hairpins. This selectivity of C. elegans HBP is mostly mediated by the first nucleotide in the loop, which is C in C. elegans and U in all other metazoans. By converting amino acids in the human RBD to the corresponding C. elegans residues at places where the latter deviates from the consensus, we could identify two amino acid segments that contribute to selectivity for the first nucleotide of the hairpin loop.