DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation.

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1-mediated histone deacetylation in heterochromatin spreading and gene silencing.

The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa. Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components. U biquitination, the addition of ubiquitin moieties to proteins, is a multistep process that regulates the intracellular stability, localization, and function of numerous proteins (1, 2). Ubiquitin is activated by an E1 enzyme and then transferred to a substrate by an E2 ubiquitin-conjugating enzyme under the direction of multisubunit cullin-RING E3 ubiquitin ligases (CRLs) (3, 4). Cullins form the scaffold of CRLs by bridging substrate adaptor proteins that interact with their N termini and catalytic proteins that interact with their C termini (4).Cullin-4 (CUL4) complexes control cell cycle progression, DNA repair, and signal transduction (5, 6). The Neurospora crassa DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for methylation of lysine 9 of histone H3 (H3K9) and DNA methylation (6). The DCDC resembles established CRLs, in which the substrate specificity adaptor protein DDB1/DIM-8 (DNA-damage-binding protein 1) serves as a bridge between CUL4 and the DCAF (DDB1 and CUL4-associated factor) DIM-9. These members of the DCDC, in turn, associate with the histone methyltransferase DIM-5 and its partner, DIM-7 (6). In addition, as in validated CRLs, CUL4 is modified by attachment of the ubiquitin-like protein NEDD8 in DCDC (6). Similarly, the Schizosaccharomyces pombe fission yeast CUL4 complex CLRC directs H3K9 methylation, although the potentially ubiquitinated substrate for this complex remains elusive (7-9), as with DCDC.Fruitless efforts to find putative substrates for ubiquitination by DCDC led us to explore the possibility that this complex has an ubiquitination-independent function. The C terminus of cullins, which is essential for the catalytic activity of CRLs, directly interacts with the E2 ligase and RBX1/ROC1, a small RING domain catalytic protein that facilitates the attachment of ubiquitin moieties onto substrates (10). The flexible backbone of cullins undergoes conformational changes to bring RBX1 and the E2 ligase in close proximity to the substrates, which are recruited by the adaptor protein bound to the N terminus (11). Covalent attachment of NEDD8 to an invariant lysine in the C terminus regulates the stability and activity of cullins (12)(13)(14)(15). To explore the contribution of neddylation in the reg...