Nucleolus and rRNA Gene Chromatin in Early Embryo Development

Abstract

The nucleolus is the largest substructure in the nucleus and forms around the nucleolar organizer regions (NORs), which comprise hundreds of rRNA genes. Recent evidence highlights further functions of the nucleolus that go beyond ribosome biogenesis. Data indicate that the nucleolus acts as a compartment for the location and regulation of repressive genomic domains and, together with the nuclear lamina, represents the hub for the organization of the inactive heterochromatin. In this review, we discuss recent findings that have revealed how nucleolar structure and rRNA gene chromatin states are regulated during early mammalian development and their contribution to the higher-order spatial organization of the genome.

Keywords: early development; genome architecture; heterochromatin; nucleolus; oocyte; rRNA genes.

Figure 1. The three major classes of rRNA genes.

(A) Structural organization of mouse rRNA gene. The sites of transcription initiation of the 45S pre-rRNA from the main gene promoter and IGS-rRNA transcripts from the spacer promoter are indicated by arrows. Terminator elements downstream of the spacer promoter (T-1), upstream of the main gene promoter (T0) and downstream of the coding regions (T1-T10) are marked by orange bars. The repeats composing the enhancer (13 according to the sequence from Genbank accession number BK000964) are shown as blue bars. ETS, external transcribed spacer. (B) Description of active, inactive and silent rRNA genes based on transcription, chromatin and epigenetic features and factors regulating their state. The composition at the main rRNA gene promoter is described. The binding of UBF and NoRC (TIP5 and SNF2H) define active and silent rRNA genes. Inactive rRNA genes are non-transcribed repeats that lack promoter DNA methylation, are nucleosome-packed at the coding region, and are not bound by UBF or NoRC. The structure of inactive genes can be mediated by NuRD, eNOSC or other yet unknown regulators. Establishment of silent rRNA genes is described. NoRC is recruited to the promoter through the interaction with TTF-1 mediated by the lncRNA pRNA. Subsequently, NoRC recruits factors that establish epigenetic silencing, such DNA methyltransferase (DNMs) histone deacetylase (HDAC1), histone methyltransferases (SETDB1), and poly(ADP-Ribose) Polymerase 1 (PARP1).

Figure 2. The nucleolar cycle in the germ line and during early development.

Illustration of mouse oogenesis and the early development timing. The nucleoli of growing oocytes in mammals are composed of fibrillar centers (red), dense fibrillar components, and granular components (purple). At the end of the growth phase, the nucleolus is transformed into nucleolus like body (NLB, black) that lacks the tripartite structure. Oocytes acquire two main types of nuclear organization: the SN-type (Surrounded Nucleolus) and the NSN-type (Non Surrounded Nucleolus). SN type is transcriptionally inactive and the NLB is surrounded by heterochromatin in a ring-like shape (grey lane). NSN type is transcriptionally active and display aggregation of heterochromatin in chromocenters (grey circles). During oocyte maturation, the nucleoli (NLBs) disappear, and upon fertilization reappear in the pronuclei of the zygote in the form of nucleolus precursor bodies (NPB, black). At the end of the 2-cell stage, transcription is initiated at the surface of NPB where UBF (green) and NoPP140 (blue) localize. From 4-cell stage, the NPB gradually transforms into somatic nucleoli that display the tripartite structure. Timing of rRNA gene transcription, nucleolus and the transition from totipotency to pluripotency are shown.

Figure 3. Establishment of silent rRNA genes during ESC differentiation.

The pluripotency state in ICM can be immortalized in vitro through culturing of embryonic stem cells (ESCs). ESCs display an open and euchromatic genome that is remodeled into a condensed and heterochromatic state, including the formation of highly compact and transcriptional repressed heterochromatic regions that cluster at the nucleolus or at the nuclear periphery. rRNA genes are all active in ESCs and establishment of silent copies occurs only upon differentiation. In ESCs, the processing of IGS-rRNA into pRNA is impaired. This process is reactivated only upon differentiation. The association of TIP5 with the unprocessed IGS-rRNA prevents the interaction with TTF-1 that is bound to the promoter, thereby abrogating rRNA gene silencing. Upon differentiation, mature pRNA is produced and promotes TIP5-TTF1 interaction that is productive for NoRC guiding to rRNA genes and formation of heterochromatin at nucleoli. The formation of silent and heterochromatic rRNA genes coincides with the remodeling of the genome from a euchromatic into a heterochromatic state that favors the exit from pluripotency.