Developmental systems and their dysfunction

A specific function performed by the brain is learning—new information is stored as short-term memory by the activation of the transcription factor CREB, and as long-term memory by DNA methylation and demethylation of specific genes. Learning also involves a neuron-specific remodeling complex (BAF) and several micro RNAs (miRNAs) and long non-coding (lncRNAs). Rubinstein–Taybi, Rett or fragile X syndromes, as well as Alzheimer’s, Parkinson’s or Huntington’s diseases, involve epigenetic alterations. Epigenetic misregulation occurs in cardiopathies such as Wolf–Hirschhorn and Kabuki syndromes. The innate immune system consists of cells that can destroy invading bacteria and virus-infected cells, and of circulating proteins that destroy pathogens. The adaptive immune system consists of macrophages and dendritic cells, T lymphocytes and B lymphocytes. Failure to recognize antigens as one’s own leads to autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Cells from RA and SLE patients exhibit changes in histone acetyl transferases, deacetylases and methyl transferases, and in miRNAs. Arginines can be converted to citrulline, and citrullinated proteins are considered as non-self by the immune system. RA is characterized by the presence of autoantibodies against citrullinated peptides.

Aging, cellular senescence and cancer: epigenetic alterations and nuclear remodeling

Epigenetic modifications correlated with aging and oncogenesis are changes in the pattern of DNA methylation and of histone modifications, and changes in the level of histone variants (H3.3, macroH2A, H2A.Z) and gene mutations. The sirtuins are a set of highly conserved protein deacetylases of particular significance to the aging process. Many cancer types are found to carry mutations in chromatin-modifying genes such as those encoding methyl or acetyl transferases, affecting the histone modifications of promoters and enhancers. The aging process and oncogenesis present a number of changes in the nuclear architecture. Mutations in the lamina-coding genes lead to premature aging syndromes. Mutations in remodeling complexes are found in different cancers. Modifications that affect the architectural protein binding sites at topologically associating domain (TAD) borders can cause the merging of neighboring TADs. The levels of short non-coding RNAs (sncRNAs) are altered in model organisms and are associated with cancer. Changes in the position of chromosome territories often occur in tumor cells. Nevertheless, cellular senescence, due mostly to the absence of telomerase, represents a mechanism of tumor suppression.

The nuclear envelope

The nuclear envelope is a double membrane sheath made up of two lipid bilayers—an outer and an inner membrane. The inner surface of the inner membrane is associated with a meshwork of filaments made up of lamins and of lamin-associated proteins that constitute the lamina. A substantial portion of the genome contacts the lamina through lamina-associated domains (LADs). LADs usually position silent or gene-poor regions of the genome near the lamina and nuclear membrane. The position of some LADs is different in some cells of the same tissue, reflecting the stochastic nature of gene activity; it can also change during differentiation, allowing the necessary activation of particular genes. Contact of transcription units with nuclear pores can result in activation or, sometimes, repression. Some of the proteins that contribute to the structure of the pores can activate transcription by associating with genes or with super-enhancers away from the nuclear membrane.

Maintenance of the active and inactive states

The maintenance of a gene in an active or inactive state is carried out by epigenetic modifications of the histones and of the DNA itself. Two major classes of complexes (PRC1 and PRC2), containing Polycomb group (PcG) proteins mediate transcriptional repression. PRC2 trimethylates histone H3 at lysine 27, a modification that attracts PRC1 leading to the ubiquitination of histone H2A. Variant PRC1 complexes can be targeted first, and mono-ubiquitinated histone H2A recruits PRC2 complexes that serve as the target for canonical PRC1 complexes. PRC2 can be targeted to sites of repression by associating with long non-coding RNAs. Trithorax group (TrxG) proteins form complexes that counteract PcG-mediated repression. Some subunits of these complexes maintain and enhance transcription by carrying out different lysine methylations (H3K4me, H3K36me and H3K79me) that are associated with active gene function; other subunits remodel chromatin by displacing and repositioning nucleosomes. Additional effects on transcription are transvections, whereby somatic pairing allows the regulatory region of one allele of a gene to influence the activity of the promoter of the allele on the homologous chromosome

Inheritance of chromatin modifications through the cell cycle

Following mitosis, the particular transcriptional landscape of the parent cell must be faithfully transmitted to daughter cells. Although transcription ceases, not all transcription factors are displaced. DNA methylation has been implicated in the inheritance of chromatin characteristics because maintenance DNA methyl transferases methylate CpG dinucleotides on the newly replicated strand if the corresponding GpC on the parent strand is methylated. Nucleosomes that are deposited on the newly synthesized DNA strands are made up of old and new histones, and some marks present on the old histones are maintained. The proper distribution of nucleosomes and the topological organization of the genome into topologically associating domains (TADs) must be transmitted to daughter cells. Following DNA replication, centromeres must be specified on the daughter chromatids. In most eukaryotes, centromeres are identified by the presence of nucleosomes bearing the histone H3 variant CENP-A. An additional number of proteins and non-coding RNAs originating from centric and pericentromeric DNA repeats associate with centromeres and appear to play a role in centromere function.

DNA repair and genomic stability

A number of pathways have evolved in order to repair DNA. Mismatch repair (MMR) operates when an improper nucleotide is used or when an insertion or deletion occurs during replication. Nucleotide excision repair (NER) repairs damage that distorts the DNA helix such as the presence of pyrimidine dimers induced by ultraviolet light. Base excision repair (BER) removes damaged or altered DNA bases that do not result in a conformational change in the chromatin. Single-strand break repair (SSBR) uses the same enzymatic steps as BER. Double-strand break (DSB) repair can involve either non-homologous end-joining (NHEJ) or homologous recombination (HR). In NHEJ, the broken DNA ends are joined directly. HR requires that one of the strands of the broken DNA molecule participates in the strand invasion of the sister chromatid. The site of the DSB must be modified to allow access to the repair machinery. This modification involves remodeling complexes, as well as histone-modifying enzymes.

The nucleolus

The nucleolus forms at nucleolus organizer regions (NORs) that consist of clusters of repeated rRNA genes. Transcription of the rRNA genes and processing of the transcripts yields the three types of RNAs necessary for the biogenesis of ribosomes. Only subsets of the rRNA genes present in cells are transcribed. The linker histone H1 plays a specific role in the repression of inactive rRNA genes and in many of the other functions of the nucleolus. One of these functions is gene silencing—the nucleolus is surrounded by a zone of heterochromatin consisting of silenced rRNA gene arrays, DNA repeats that flank the centromeres and chromatin domains that include gene-poor, as well as silent, regions of the genome; any gene associating with this zone is subjected to repression. Other functions include the assembly of telomerase, the regulation of p53 stability and the synthesis of 5S and tRNAs whose genes form clusters in the nucleolus.

The basic mechanism of gene transcription

Transcription is initiated by factors that interact with RNA polymerases and recruit them to specific sites, unwind the DNA molecules and allow the synthesis of RNA transcripts complementary to one of the single DNA strands. RNA polymerase II (RNAPII) transcribes genes that encode proteins and some non-coding RNAs; RNAPI transcribes ribosomal RNA genes; RNAPIII transcribes genes that encode tRNAs and other non-coding RNAs. The transcription process starts with a pre-initiation complex (PIC), its activation and promoter clearance. Activation involves chromatin looping, usually promoted by the large multiprotein Mediator complex. RNAPII often makes a promoter-proximal pause, then resumes productive elongation of the transcript. Transition through the different phases of transcription is orchestrated by the phosphorylation of the main subunit of RNAPII. The 5´ end of many transcripts is protected by a methylated guanosine “cap,” and the 3´ end by the addition of a chain of adenosine monophosphates (polyadenylation). Many transcripts undergo splicing to remove regions that interrupt the coding sequence.

The basic structure of chromatin

In cells, DNA is associated with histones, non-histone proteins and RNA in a complex referred to as chromatin. Four different types of histones form octamers (nucleosomes), around which DNA is wrapped yielding a chromatin fiber with the configuration of “beads on a string.” Disassembly, followed by reassembly, of this structure occurs during DNA replication, damage repair and transcription. Core histones are replication-coupled; variants are replication-independent. Positioning of nucleosomes on the chromatin fiber is mediated by chromatin remodeling complexes and reflects the functional state of various regions along the fiber. Various biophysical methods have been utilized to study the physical association of nucleosomes and DNA. Chromatin can be differentiated on the basis of the activity of the genes that are present in a given region. Heterochromatin represents repressed or inactive regions of the genome and exhibits a greater degree of condensation than euchromatin, which refers to more unwound regions where active genes are located. The two types of chromatin are present in different nuclear locations.

Nuclear reprogramming and induced pluripotency

Four core transcription factors known to maintain the pluripotent state in embryonic stem cells (ESCs)—Oct4, Sox2, Klf4 and c-Myc—were used to induce pluripotent stem cells in adult-derived fibroblasts. Induced pluripotent stem cells (iPSCs), like ESCs, have less condensed and more transcriptionally active chromatin than differentiated cells. The number of genes with bivalent promoter marks increases during reprogramming, reflecting the switch of differentiation-specific active genes to an inactive, but poised, status. The levels of DNA methyl transferases and demethylases are increased, underlying the changes in the pattern of DNA methylation that occur late during reprogramming. The potential therapeutic applications of iPSCs include reprogramming a patient’s own cells to avoid the problem of rejection following injection to restore tissue or organ function. iPSCs derived from individuals at risk of developing late-onset neurological diseases could be differentiated in culture to predict the future occurrence of the disease. Caveats involve the fact that long-term culturing often results in genomic mutations that may, by chance, involve tumor suppressors or oncogenes.