Classification and monomer-by-monomer annotation dataset of suprachromosomal family 1 alpha satellite higher-order repeats in hg38 human genome assembly.

In the latest hg38 human genome assembly, centromeric gaps has been filled in by alpha satellite (AS) reference models (RMs) which are statistical representations of homogeneous higher-order repeat (HOR) arrays that make up the bulk of the centromeric regions. We analyzed these models to compose an atlas of human AS HORs where each monomer of a HOR was represented by a number of its polymorphic sequence variants. We combined these data and HMMER sequence analysis platform to annotate AS HORs in the assembly. This led to discovery of a new type of low copy number highly divergent HORs which were not represented by RMs. These were included in the dataset. The annotation can be viewed as UCSC Genome Browser custom track (the HOR-track) and used together with our previous annotation of AS suprachromosomal families (SFs) in the same assembly, where each AS monomer can be viewed in its genomic context together with its classification into one of the 5 major SFs (the SF-track). To catalog the diversity of AS HORs in the human genome we introduced a new naming system. Each HOR received a name which showed its SF, chromosomal location and index number. Here we present the first installment of the HOR-track covering only the 17 HORs that belong to SF1 which forms live functional centromeres in chromosomes 1, 3, 5, 6, 7, 10, 12, 16 and 19 and also a large number of minor dead HOR domains, both homogeneous and divergent. Monomer-by-monomer HOR annotation used for this dataset as opposed to annotation of whole HOR repeats provides for mapping and quantification of various structural variants of AS HORs which can be used to collect data on inter-individual polymorphism of AS.

Annotation of suprachromosomal families reveals uncommon types of alpha satellite organization in pericentromeric regions of hg38 human genome assembly.

Centromeric alpha satellite (AS) is composed of highly identical higher-order DNA repetitive sequences, which make the standard assembly process impossible. Because of this the AS repeats were severely underrepresented in previous versions of the human genome assembly showing large centromeric gaps. The latest hg38 assembly (GCA_000001405.15) employed a novel method of approximate representation of these sequences using AS reference models to fill the gaps. Therefore, a lot more of assembled AS became available for genomic analysis. We used the PERCON program previously described by us to annotate various suprachromosomal families (SFs) of AS in the hg38 assembly and presented the results of our primary analysis as an easy-to-read track for the UCSC Genome Browser. The monomeric classes, characteristic of the five known SFs, were color-coded, which allowed quick visual assessment of AS composition in whole multi-megabase centromeres down to each individual AS monomer. Such comprehensive annotation of AS in the human genome assembly was performed for the first time. It showed the expected prevalence of the known major types of AS organization characteristic of the five established SFs. Also, some less common types of AS arrays were identified, such as pure R2 domains in SF5, apparent J/R and D/R mixes in SF1 and SF2, and several different SF4 higher-order repeats among reference models and in regular contigs. No new SFs or large unclassed AS domains were discovered. The dataset reveals the architecture of human centromeres and allows classification of AS sequence reads by alignment to the annotated hg38 assembly. The data were deposited here: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&hgt.customText=https://dl.dropboxusercontent.com/u/22994534/AS-tracks/human-GRC-hg38-M1SFs.bed.bz2.

Regulation of actin cytoskeleton in lymphocytes: PKC-delta disrupts IL-3-induced membrane ruffles downstream of Rac1.

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL-3. Constitutively activated Rac1 restores the elongated cell phenotype to IL-3-starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL-3-induced assembly of membrane ruffles. Similar to the IL-3 withdrawal, phorbol 12-myristate 13-acetate (PMA) dissolves actin-formed membrane ruffles and rounds the cells in the presence of IL-3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL-3 the PMA-induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL-3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA-induced cell rounding can be reversed by the PKC-specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC-delta indicate that activation of PKC-delta but not other PKC isoforms is responsible for disruption of membrane ruffles.

Cross-talk between protein kinase C-alpha (PKC-alpha) and -delta (PKC-delta): PKC-alpha elevates the PKC-delta protein level, altering its mRNA transcription and degradation.

Studies utilizing the overexpression of individual isoforms indicated that both PKC-alpha and -delta promote a number of biological effects, including inhibition of DNA synthesis associated with rearrangements of the actin cytoskeleton in the murine B-cell lymphoma (Baf3), differentiation of the murine promyelocyte line 32D, and activation of MAP kinase in CHO fibroblasts. We postulated that these results reflect some form of cross-regulation between PKC-alpha and -delta rather than their functional redundancy. In this report, we show that overexpression of PKC-alpha in Baf3 and 32D leads to an elevation of the endogenous PKC-delta mRNA and protein levels. The elevated steady-state PKC-delta mRNA level results from a combination of increased PKC-delta transcription and mRNA stability. Upregulation of PKC-delta mRNA by PKC-alpha occurs even after a selective depletion of the PKC-delta protein. In addition, phorbol ester-induced elevation of PKC-delta mRNA and protein levels can be prevented by the PKC inhibitor GF109203X, an indication of the requirement for PKC kinase activity. Inhibition of new protein synthesis by cycloheximide showed that upregulation of PKC-delta mRNA, as opposed to delayed downregulation of the PKC-delta protein, is primarily responsible for the accumulation of this isoform by PKC-alpha. In parental Baf3 and 32D cells and PKC-alpha overexpressers, PKC-alpha and PKC-delta are uniquely involved in cross-regulation, while PKC-epsilon, PKC-eta, and PKC-mu are not.

Evidence for selection in evolution of alpha satellite DNA: the central role of CENP-B/pJ alpha binding region.

Conservation of DNA segments performing sequence-related functions is a landmark of selection and functional significance. Phylogenetic variability of alpha satellite and apparent absence of conserved regions calls its functional significance into question, even though sequence-specific alpha satellite-binding proteins pJ alpha and CENP-B have been discovered. Moreover, the function of pJ alpha is obscure and CENP-B binding satellite DNA, which is thought to participate in centromere formation, is found only in few species and not necessarily in all chromosomes. Analysis of alpha satellite evolution allows us to recognize the order in this variability. Here we report a new alpha satellite suprachromosomal family, which together with the four defined earlier, covers all known alpha satellite sequences. Although each family has its characteristic types of monomers, they all descend from two prototypes, A and B. We show that most differences between prototypes are concentrated in a short region (positions 35 to 51), which exists in two alternative states: it matches a binding site for pJ alpha in type A and the one for CENP-B in type B. Lower primates have only type A monomers whereas great apes have both A and B. The new family is formed by monomeric types almost identical to A and B prototypes, thus representing a living relic of alpha satellite. Analysis of these data shows that selection-driven evolution, rather than random fixation of mutations, formed the distinction between A and B types. To our knowledge, this is the first evidence for selection in any of the known satellite DNAs.

Mechanism of apoptosis suppression by phorbol ester in IL-6-starved murine plasmacytomas: role of PKC modulation and cell cycle.

We show here that the mode of cell death in IL-6-starved T1165 and T1198 plasmacytoma cell lines is apoptosis, and that it can be suppressed by phorbol ester (PMA) treatment in a protein kinase C (PKC)-mediated process that involves alpha and/or delta isozymes. PMA-induced PKC activation, but not the depletion that follows it, participates in the suppression of apoptosis. Extended PKC activation is necessary but not sufficient for the apoptosis suppression. In addition, the cells must be in a "competent" state, which appears not to be determined by PKC. We observed two points of "competence" during the time between withdrawal of IL-6 and the start of massive cell death: one, immediately after withdrawal, and another, just before onset of apoptosis, at the time corresponding to maximal accumulation of cells in a G0/G1 block imposed by IL-6 withdrawal. Treatment with PMA and other PKC activators resulted in a shift of the cell population to S phase, lifting the G0/G1 block. We propose a model in which cells are rescued in a certain stage of the G1 phase of cell cycle. Death suppression occurs when a transient PMA-induced PKC activation occurs when a significant number of cells are in this part of G1, allowing them to pass the restriction point safely without initiating the cell death program.

Genomic organization, sequence and polymorphism of the human chromosome 4-specific alpha-satellite DNA.

Two alpha-satellite fragments specific for human chromosome 4 have been cloned and characterized. Under stringent annealing conditions, they hybridized in situ only to the pericentromeric region of chromosome 4, but under non-stringent conditions they hybridized to all chromosomes containing the sequences of alpha-satellite suprachromosomal family 2 (viz., chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21 and 22). Southern blot analysis reveals the 3.2-kb higher-order repeated unit which exists in two forms: as a single MspI fragment or a combination of the 2.6-kb and 0.6-kb MspI fragments. The two chromosome-4-specific cloned sequences appear to be different parts of this repeated unit. Taken together they constitute about 60% of its length. The primary structure of the higher-order repeated unit is characterized by a dimeric periodicity of the D1-D2 type which is usual to suprachromosomal family 2. At least in one site this regularity is disrupted by monomer deletion leading to the D2-D2 monomeric order. The most likely mechanism of this monomer excision is homologous unequal crossing-over. These sequences may serve as both cytogenetic and restriction-fragment length polymorphism (RFLP) markers for the pericentromeric region of chromosome 4.

Segment substitutions in alpha satellite DNA. Unusual structure of human chromosome 3-specific alpha satellite repeat unit.

We have sequenced the full-length copy of the alpha satellite higher-order repeated unit characteristic of human chromosome 3. Its internal structure, the regular alterations of J1 and J2 type monomers, is typical of the alphoid suprachromosomal family 1. This dimeric order is disrupted by the substitution of one J1 unit by an alien dimer which is not clearly related to any of the established monomeric types. We have also observed some other similar cases of segment substitutions in alpha satellite DNA. They probably represent a special type of molecular event which could be generated by gene conversion. Segment substitutions may be one of the important factors responsible for the extreme variability of localization patterns and actual sequences of alpha satellite DNA that should be taken into account in reconstructions of alpha satellite evolution.

Definition of a new alpha satellite suprachromosomal family characterized by monomeric organization.

We have analyzed more than 500 alphoid monomers either sequenced in our laboratory or available in the literature. Most of them belonged to the well studied suprachromosomal families 1, 2 and 3 characterized by dimeric (1 and 2) and pentameric (3) ancestral periodicities. The sequences that did not belong to the previously known families were subjected to further analysis. About a half of them formed a relatively homogenous family. Its members were on average 80.5% identical and 89.5% homologous to the M1 consensus sequence derived from this group (39 monomers). In the genome they do not form any ancestral periodicities other than a monomeric one, and are found at least in chromosomes 13, 14, 15, 21, 22 and Y. The newly defined family was termed suprachromosomal family 4. Comparison of all 10 alphoid monomeric groups identified so far showed that the M1 sequence is closely related to the J1-D2-W4-W5 homology grouping. Notably the African Green Monkey alpha satellite, also characterized by monomeric construction, appears to be a member of the same group.

Chromosome-specific alpha satellites: two distinct families on human chromosome 18.

Two types of human chromosome 18-specific alpha satellite fragments have been cloned and sequenced. They represent closely related but distinct alphoid families formed by two different types of the higher-order repeated units (1360-bp EcoRI and 1700-bp HindIII fragments) that do not alternate in the genome. The individual repeats within each family are 99% identical and interfamily homology is about 78%. Sequence analysis shows that both repeats belong to alphoid suprachromosomal family 2, but their homology is not higher than that of family members located on different chromosomes. Therefore, the two repeats shared a common origin in the recent past, although they are not the direct offspring of one ancestral sequence. Our data indicate that these two 18-specific domains have appeared as a result of two separate amplification events. Despite the high degree of homology, they are not undergoing intrachromosomal homogenization, although some variation of this process might take place within each domain.

The phylogeny of human chromosome specific alpha satellites.

The chromosomal distribution of sequences homologous to 18 coned alpha satellite fragments was established by in situ hybridization. It appeared that all the cloned sequences were members of small repeated families located on single chromosome pairs. Among the sequences studied specific molecular markers for chromosomes 3, 4, 10, 11, 17, 18 and X were found. Comparison of the hybridization spectra obtained under non-stringent conditions and of restriction site periodicities in different chromosome-specific families allowed the identification of three "suprachromosomal" families, each located on a characteristic set of chromosomes. The three families together cover all the autosomes and the X chromosome. These data plus those reported previously allow part of the phylogenetic tree of chromosome-specific alpha satellite repeats to be drawn. Each suprachromosomal family has presumably originated from a distinct ancestral sequence and consists of certain types of monomers. Ancestral sequences have evolved into a number of chromosome-specific families by cycles of interchromosomal transfers and subsequent amplification events. The high homogeneity of chromosome-specific families may be a result of intrachromosomal homogenization of amplification units in chromosome-specific alpha satellite domains.

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man.

The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.

18p- syndrome: an unusual case and diagnosis by in situ hybridization with chromosome 18-specific alphoid DNA sequence.

A patient with an atypical clinical picture of 18p- syndrome is described. By the in situ hybridization technique we localized the chromosome 18-specific cloned repetitive sequence to metaphase chromosomes of the patient. The predominant hybridization of the probe was found in pericentromeric regions of homologous chromosomes 18. The amount of pericentromeric DNA measured by in situ hybridization differed between homologous chromosomes; and the number of radioactive grains was statistically greater in the normal chromosome 18 than in the aberrant chromosome 18p-. The results indicate that this probe may be useful in clinical cytogenetics for identification of aberrant chromosomes, localization of breakpoints, and studies of C-band DNA polymorphism of chromosome 18.