**2. Physiologic Roles of Epigenetics**

During development, stable and heritable mechanisms, such as histone modifications and DNA methylation, are employed for the functional regulation of gene expression [1,2]. These processes are referred to as "epigenetics", indicating changes occurring without a direct alteration of the DNA sequence. Under this term are included all the changes exerted via the regulation of chromatin functions and states of activation that are critical for the control of DNA accessibility and transcription. Indeed, the three-dimensional folding of the nuclear genome is tightly linked to the functional DNA-dependent processes of

**Citation:** Romano, R.; Cillo, F.;

Moracas, C.; Pignata, L.; Nannola, C.; Toriello, E.; De Rosa, A.; Cirillo, E.; Coppola, E.; Giardino, G.; et al. Epigenetic Alterations in Inborn Errors of Immunity. *J. Clin. Med.* **2022**, *11*, 1261. https://doi.org/10.3390/ jcm11051261

Academic Editors: Rita Consolini and Giorgio Costagliola

Received: 20 January 2022 Accepted: 24 February 2022 Published: 25 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

replication and transcription. In particular, DNA replication is a complex and dynamic phenomenon based on the interplay among the epigenetic signature, the transcriptional activity and the structure of chromatin into which DNA is folded and condensed into the nucleus [3]. The double-stranded DNA is wrapped around a core of 8 histone proteins, including two copies of each histone, H2A, H2B, H3, and H4, forming superordinate biomolecular structures, namely nucleosomes, that build up chromatin fibers [4] (Figure 1A). Because of the flexibility of chromatin fibers, target DNA sequences can contact their regulatory elements, even though they are distantly located. Chromosomes segregate into two mutually exclusive types of chromatin, "A" and "B" compartments, including gene-rich active and repressive chromatin, respectively [5,6]. The A compartment is located centrally, whereas the B compartment is typically located peripherally in the nucleus [6]. Besides chromatin structure remodeling, DNA methylation, histone post-translational modifications and non-coding RNAs (ncRNAs) are the key epigenetic factors involved in the dynamics of transcriptional control.

**Figure 1.** (**A**) Chromosomes formed by chromatin fibers organized into nucleosomes, in which they are wrapped around eight histone proteins (as shown in the red box, zooming in chromosome structure). On a deeper level, DNA methyltransferases (DNMTs) act on double-stranded DNA, adding a methyl group to the carbon 5 (5meC) of cytosine-followed-by-guanine dinucleotides, while Ten-Eleven Translocation (TET) enzymes are responsible for demethylation, removing a 5meC (red X in the figure). (**B**) The differentiation from a common progenitor, a hematopoietic stem cell, to lymphoid and myeloid lineage, is accompanied by a wave of increasing methylation or demethylation, respectively. DNMTs: DNA methyltransferases, TET: Ten-Eleven Translocation; HSC: hematopoietic stem cell, CLP: common lymphoid progenitor, CMP: common myeloid progenitor, PMN: polymorphonucleate.

For DNA methylation, a methyl group is added to the carbon 5 (5meC) of cytosinefollowed-by-guanine dinucleotides (CG or CpG sites) by DNA methyltransferases (DN-MTs), a family of four enzymes. Unlike in mammals, bacterial methylation may also occur

at N4 cytosine (4meC) and N6 adenine (6meA), the latter being the most prevalent in bacteria [7].

In mammals, DNMT3A, DNMT3B and DNMT3L catalyze de novo DNA methylation with differential kinetics and patterns during male and female gametogenesis and within cell lineage specification in post-implantation development [8,9], while DNMT1 secures the maintenance of DNA methylation following replication through cell division [10].

In the primordial germ cells and pre-implantation embryo, two waves of extensive erasure involving both passive and active mechanisms occur. Consecutive cell divisions may be followed by passive DNA demethylation, while enzyme members of the Ten-Eleven Translocation (TET) family mediate active demethylation through the oxidation of 5-methylcytosine (5mC) to 5-formylcytosine (5fC), 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC), followed by the replication-dependent dilution of oxidized 5mC or the thymine DNA glycosylase (TDG)-mediated excision of 5fC and 5caC coupled with base excision repair [10].

In general, DNA methylation is high across gene bodies and inter-genic regions, and low at regulatory regions, such as promoters and enhancers. Once established on regulatory regions, methylation can be repressive for transcription because it either directly inhibits the binding of transcription factors or indirectly inhibits the activity of methyl-binding proteins and chromatin modifiers. The methylation of gene bodies is not a repressive mark, but it prevents spurious transcription initiation [11].

Although it is prevalent in mammals, it is worth mentioning that CpG methylation is not the only model of methylation: evidence of non-CpG methylation sites has been found in human embryonic stem cells, induced pluripotent stem cells and brain tissues [12,13]. Hence, novel research to ascertain its role in the maintenance of pluripotency, as well as in the pathophysiology of cancer and neurodegeneration, has developed significantly in recent years [14–16].

Histone marks, such as histone H3 modifications, correlate with gene expression. For example, histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 acetylation (H3K27ac) are active marks, found at active promoters and/or enhancers. They correlate negatively with DNA methylation and positively with gene expression [17].

Proper DNA methylation is required for normal human development [18]. Methylation abnormalities may be associated with genetic defects involving *cis*-acting elements or*trans*-acting factors, but can also occur in the absence of obvious genetic changes as primary epimutations; these may represent stochastic or environment-driven errors in the establishment or maintenance of an epigenetic program [19]. Single-locus methylation defects can be a consequence of a variant occurring in *cis*, while, when multiple loci are involved, this may be due to a variant occurring in *trans*. In the latter case, variants in DNMTs or mutations in chromatin modifiers or transcription factors may alter genomic methylation [18].

In cancer, abnormal DNA methylation patterns have been frequently demonstrated, such as the hypermethylation of tumor suppressor gene promoters or the methylation changes of imprinted loci [20].

Recently, through the use of high-throughput screening platforms, an increasing number of disorders have been associated with specific "episignatures", indicating that DNA methylation analysis may represent a powerful tool for the more accurate classification of diseases with overlapping clinical signs and for categorizing cases with unclear genetic variants [21].

The so-called non-coding RNAs include microRNAs (mRNAs) and long-non-coding RNAs (lncRNAs), both of which are involved in the regulation of gene expression. The former are short molecules that bind to complementary sequences in the 3 UTR region of the mRNA, directly inhibiting its translation or inducing its degradation. The latter are longer than 200 nucleotides and act by binding to histone modifiers or transcription regulation proteins [22]

Since they play a crucial role in regulating processes such as proliferation, differentiation, development, and apoptosis, it is not surprising that the disruption of their

function is also relevant for human diseases, as uncovered by evidence gained in the field of tumorigenesis [23].
