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Transcriptional activation of specific genes is initiated after fertilization by the interaction of specific transcription factors with its cognate sequences in the chromatin context, thereby leading to a concerted and coordinated program which determines early development. Remodeling of the sperm chromatin after fertilization is a fundamental event for transcriptional activation and expression of the paternally inherited genome. The transitions in chromosomal proteins, as well as the mechanisms that participate in these transitions, have been investigated only to a limited extent as compared to the signal transduction patterns that govern egg activation or the dynamics and structural changes accompanying sperm nuclear membrane dissociation-association following insemination. In this review, we will discuss the remodeling of sperm chromatin that follows fertilization. We will emphasize the transitions of chromosomal proteins, as well as the post-translational modifications associated with these transitions. The molecular mechanisms that may be participating in these events will also be analyzed. We will further discuss the mechanisms that govern chromatin remodeling and the role of specific transcription factors in the control of the transcriptional program during sea urchin early development.

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization., (Copyright 2002 Wiley-Liss, Inc.)

During sea urchin embryogenesis the early histone genes are temporally expressed to accommodate the high demand for histone proteins during DNA replication at early cleavage stages of development. The early histone genes are transcriptionally active from the 16-cell stage, reaching a peak in expression at the 128-cell stage that gradually decreases until expression is completely inhibited at the late blastula stage. We are studying the gene regulatory mechanisms that control early histone gene expression in sea urchins to understand the interrelationships between chromatin remodeling and transcriptional activation during development. Here, we have investigated chromatin organization and transcription factor interactions by analyzing nuclease hypersensitivity and protein binding in the promoter region of the early histone H3 gene from the sea urchin Tetrapygus niger. We have found a DNase I hypersensitive domain centered at -90 in the early histone H3 gene promoter which is only detected in embryos at the 128-cell stage expressing high levels of early histone H3 mRNA. This hypersensitive site (-110 to -70) encompasses two regulatory elements (TnH3NFH3.1 and TnH3CCAAT). The -94 to -77 region of the histone H3 promoter is recognized by a transcription factor complex in nuclear extracts from 128-cell embryos. Methylation interference analysis and competition studies demonstrated a specific interaction at the CCAAT sequence. Using specific antibodies we find that the homeodomain transcription factor CDP/cut is the DNA-binding component of the complex interacting with the early histone H3 gene promoter in T. niger. Our results provide further evidence for the functional role of CDP/cut in developmental regulation of histone gene expression in phylogenetically diverse eukaryotic species.

To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants.

After fertilization the cone shaped sperm nucleus is transformed into a spheroidal male pronucleus which fuses with the female pronucleus reestablishing the diploid genome. These morphological changes are correlated with biochemical transitions of sperm-specific chromosomal proteins. To obtain information on the rearrangements of chromosomal proteins after fertilization, we have followed sperm-specific nonhistones (Sp NHCP) and the sperm-specific histones (SpH) in zygotes harvested at different times post-insemination (p.i.). The acquisition of proteins synthesized de novo was determined during the time of male pronucleus formation, estimated to occur until 30 min p.i. The results obtained may be summarized as follows: 1. The fate of Sp NHCP, followed by Western blot analysis with polyclonal antibodies obtained in rabbits against the whole complement of Sp NHCP, indicates that the majority of Sp NHCP are lost shortly p.i., except two proteins of 58 Kd and 61 Kd that were not distinguishable from eggs NHCP because a crossreaction was obtained. Acidic proteins synthesized de novo were bound to chromatin between 3 and 30 min p.i., and the majority of these proteins comigrated with egg NHCP in SDS/PAGE. 2. The histones from sperm differ from their counterparts found in unfertilized eggs, both in their amino acid composition and their microheterogeneity in bidimensional gels. Sperm contain the five major SpH whereas eggs have seven major histone variants, distinct from each other, with an electrophoretic behaviour consistent with histones but a different amino acid composition. The total complement of histone variants found in zygotes collected at amphimixis is identical with that found in unfertilized eggs, suggesting that SpH should be lost before that time.(ABSTRACT TRUNCATED AT 250 WORDS)

A close relationship between histone synthesis and DNA replication has been suggested for many biological systems. The presence of histonic proteins has been demonstrated in practically all kinds of eukaryotic cells. During the clevage period of sea urchins it has been postulated that histones are not present in nuclei, and that only at blastula stage they do associate with DNA. Our results suggest that the failure to isolate histonic proteins from nuclei derived from cleavage cells might be caused by the presence in those cells of proteases activated by NaHSO3. This compound has being widely used to inhibit proteolytic action in other biological systems. By changing the method for chromatin isolation, we have been able to isolate basic proteins from nuclei of gametes, zygotes and 2-4 blastomeres. These proteins behave like calf thymus histones in urea-acetic acid polyacrylamide gels, and they do not show great differences from proteins isolated from nuclei of blastula, gastrula, prism, and pluteus. The electrophoretic patterns of basic proteins obtained from eggs and zygotes are practically identical, except for one protein moving like lysine-rich calf thymus histones. This protein appears in zygote nuclei at the beginning of the first replication wave.