“Memory” lies at the heart of cellular identity, underscoring the existence of fundamental mechanisms to ensure that cells remember who they are and how they move along elaborate pathways of cellular differentiation, a cornerstone of multicellular organisms.
During development, germ cells or totipotent stem cells give rise to a diverse array of specialized cell types. Cells can be significantly affected by insults, injury and changes in metabolism.
This calls into place a poorly understood “reprogramming process” that may be able to erase previously established settings and, possibly, dedifferentiate or revert these cells to a more primitive pluripotent state. Thus, it is reasonable to note that the developmental process requires “forward” differentiation with a built-in memory component as well as a “reversible” reprogramming capability, allowing for plasticity.
How could one relatively fixed genome permit this level of flexibility? There must be a system that allows a constant genetic blueprint to be organized in such a way as to accommodate variability resulting from extrinsic and intrinsic signals that come from environmental, dietary and other influences.
A wealth of recent work from many laboratories has rekindled an interest in an old word — epigenetics. It is fitting that epigenetics, as well as the general concept of an “epigenetic landscape,” was first articulated by a developmental biologist, Conrad Waddington, who used the word to explain how identical genotypes could unfold a wide collection of phenotypes as development proceeded. With time, Waddington’s concept of a phenotypic landscape took on additional meaning — “potentially heritable changes in gene expression that do not involve changes in DNA sequence.” In today’s biology, epigenetics occupies a central position, jump-started by the sequencing of the human genome and reinforced with clear links to human biology and disease, notably cancer.
While remarkable advances have been made during the past two decades, to date it still unclear how these dedicated machines are directed to or guided to their target sequences, but it is likely to involve constellations of cis-acting regulatory proteins and non-coding RNAs that engage the DNA template directly.
Waddington’s landscape has taken a clearer form with the documentation of a remarkable variety of molecular pathways responsible for epigenetic control in all cells. These include multi-subunit complexes that act to remodel chromatin — to exchange specific histones (histone variants) in and out of assembled chromatin — or to enzymatically modify DNA and histone proteins to bring about downstream events.
Epigenetic control may be exerted through a variety of mechanisms, including: DNA methylation, miRNA metabolic pathways,histone variants, post-translational mechanisms.The interplay of these regulatory mechanisms suggests that the coordinate and progressive combination of these processes may allow the epigenome to move from an “unlocked” to a “locked” state, thereby determining the fate and physiology of a given cell.
Histone PTMs are responsible in large part for the plasticity of chromatin remodeling. Histone modifications are highly dynamic and often reversible events that allow cells to modify their gene transcription, depending on environmental changes. These events require recruitment of nuclear remodeling factors, often organized in large protein complexes, to induce chromatin transitions and permit or inhibit the access to DNA by transcription factors. Enzymes that modify histone tails, sometimes contained in these complexes as subunits, are brought to a specific region of DNA by DNA-binding proteins and they can either facilitate the access to the DNA or cause further compaction, depending on the tail modification. Histones are modified at multiple amino acid residues in more than 30 sites within their N-terminal tails. Many different post-translational modifications (acetylation, methylation, phosphorylation, sumoylation, ubiquitination, ADP-ribosylation and biotinylation) can occur, each elicited by specific chromatin remodeling enzymes.
An important consideration relates to the intracellular pathways involved in the marking of these PTMs. Importantly, all of them use metabolites, thereby indicating that the dynamic process of chromatin remodeling “senses” cellular metabolism and changes in energy levels, which are highly controlled and functionally essential in all physiological responses. One example in which chromatin remodeling directly influences mammalian physiology is the circadian clock, through which at least 15 percent of the genome is controlled. Circadian rhythms govern a wide variety of metabolic functions in most organisms. At the heart of these regulatory pathways is the clock machinery, a remarkably coordinated transcription-translation system that utilizes dynamic changes in chromatin states.
The exquisite control of circadian gene expression by the clock is associated to chromatin remodeling. The very first observation of circadian chromatin transitions illustrated that H3-Ser10 phosphorylation occurs in SCN neurons in response to a light stimulus and is linked to the activation of clock genes (Crosio et al. 2000). Subsequently, a number of studies have described chromatin remodeling on circadian genes and identified some histone modifying enzymes to be involved in circadian activity (Koike et al. 2012; Papazyan et al. 2016). We have found that the circadian regulator CLOCK has histone acetyltransferase (HAT) activity (Doi et al. 2006). CLOCK acetylates histone H3 at Lys 9/14, as well as its partner BMAL1, an event that leads to circadian control of gene expression (Hirayama et al. 2007). Importantly, additional results demonstrate that CLOCK acetylates also cytoplasmic proteins, such as argininosuccinate synthase 1, an enzyme that catalyzes the penultimate step of the arginine biosynthetic pathway (Lin et al 2017). This feature of CLOCK establishes important connections to a number of cellular functions and pathologies.Among the chromatin remodelers involved in circadian control, the nicotinamide adenine dinucleotide (NAD+)-dependent SIRT1 deacetylase deserves special mention. Indeed, SIRT1 and other members of the so-called ‘sirtuin’ family provide a relevant molecular link between metabolism, epigenetics and the circadian clock (Nakahata et al. 2008).
SIRT1 is an enzyme that has been shown to regulate aging, inflammation and metabolism. Strikingly, SIRT1 activity is NAD+-dependent, directly linking cellular energy to epigenetics. Additional findings indicate that regulation also goes the other way, since specific elements of the clock are able to sense changes in the cellular metabolism. Our laboratory has also demonstrated that other nuclear chromatin remodelers are involved in circadian gene expression. These include SIRT6, another member of the sirtuin family. SIRT6 has the characteristic of being prominently nuclear and chromatin bound. We have discovered that the two sirtuins SIRT1 and SIRT6 contribute in partitioning the epigenome with the consequence of each directing distinct metabolic pathways (Masri et al. 2014).
Further studies have uncovered the role of the H3K4 tri-methyltransferase MLL1 in circadian control. MLL1 interacts with CLOCK and BMAL1 and is essential for their recruiting to chromatin at clock-driven gene promoters (Katada and Sassone-Corsi 2010). Remarkably, MLL1 is acetylated in a circadian manner at specific lysine residues which are deacetylated by SIRT1, thereby linking NAD+-dependent metabolism with histone methylation (Aguilar-Arnal et al. 2015).
By applying 4C technology, our laboratory discovered that the circadian clock directs spatial and temporal changes in nuclear organization which lead to cyclic gene expression. This finding underscores the pervasive nature of circadian clock control at the molecular level and paves the way to further explore how spatial nuclear organization may explain how the clock controls different groups of genes in various cell types (Aguilar-Arnal et al. 2013).
Understanding in full detail the intimate links between cellular metabolism and epigenetics will provide not only critical insights into system physiology, but also novel avenues toward the pharmacological intervention of metabolic disorders.
Chromatin Remodeling in Male Germ Cells
The program of gene expression that characterizes male germ cells is unique for several reasons. First, in spermiogenesis cells are haploid; second, the chromatin structure is completely different from all somatic cells; third, a collection of unique transcription factors is used in this differentiation program. In a series of studies spanning more than 15 years, we elucidated the molecular and endocrine mechanisms that lead to the activation of the male germ-specific transcription program, including specific isoforms of the CREM protein and several other regulators. These studies allowed for the deciphering of a specific protein complex that directs post-meiotic transcription as well as the identification of novel molecular pathways including: 1) The discovery of a kinesin motor protein in the nuclear transport of a transcriptional co-activator; 2) The discovery of the chromatoid body as an RNA-processing organelle; 3) The discovery of several germ cell-specific transcription factors and histone variants. 4) Uncovering the unique epigenetic program of male germ cells. The impact of these studies is extensive in the fields of transcriptional regulation, endocrinology and reproduction.
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Editors: Allis, Caparros, Jenuwein, Reinberg, and Lachlan