Precise Switching of DNA Replication Timing in the GC Content Transition Area in the Human Major Histocompatibility Complex

Abstract

The human genome is composed of long-range G؉C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.The genomes of warm-blooded vertebrates, including humans, are composed of large-scale compartmentalized structures, one of which consists of long-range GϩC% (GC%) mosaic structures. The mosaics of long-range regions (Ͼ300 kb) homogeneous in GC% are called isochores by Bernardi et al. (5). Several groups, including ours, showed that the GC% mosaic structures are related to chromosome bands (1,5,23,28,29,31,44). Giemsa-dark G bands are composed mainly of AT-rich sequences, whereas T bands (a subgroup of Giemsapale R bands) are composed mainly of GC-rich sequences: ordinary R bands are heterogeneous and appear to be intermediate in GϩC content (3,4,12,(32)(33)(34)47). Another aspect of genome compartmentalization is the replication time zone. Mammalian DNA replication timing is roughly divided into early S (S E ) and late S (S L ) phases (16, 25, 38); G-band zones replicate primarily during S L , whereas R-band zones, including T bands, replicate in S E (27). Therefore, band boundaries have been presumed to be precisely assignable at the nucleotide sequence level by identifying the early-to-late switch point for replication timing. We would predict that this region is where regulatory signals for DNA replication exist. Band boundaries should also be suitable for studying the mechanisms involved in termination, pausing, and/or slowing of DNA replication fork movement.The human major histocompatibility complex (MHC) is known at a high-resolution level to be composed of plural bands (49, 53...

GC/AT-content spikes as genomic punctuation marks

“…We suggest that the GC-content spikes may represent important compositional factors that define different functional genomic units, particularly transcription boundaries. Regions with sharp GC-content changes should be structurally different from the rest of the genome, where the GC content is constant or gradually changing and thus should be recognizable from the rest of genome (9,22,23). Therefore, GC-content spikes may play a general role in delineating different functional regions of the genome.…”

Replication Timing Profile Reflects the Distinct Functional and Genomic Features of the MHC Class II Region

“…Sharp transitions to earlier replication timing are seen on either side of the later replicating domains. The transition at the class II/III boundary has been reported as a replication "switch" region in HL60 cells, 23 and is at the site of a pronounced shift in GC content. The earliest replication time is observed in the class III region close to the HSPA1 gene cluster where a replication origin has been identified.…”

Genomic Anatomy of a Premier Major Histocompatibility Complex Paralogous Region on Chromosome 1q21–q22

“…2F;Bernardi 1995). This G + C content is, however, much lower than the densely packed 6p HLA region, which belongs to the G + C-rich isochore H1 (46.2%) (Fukagawa et al 1995;Tenzen et al 1997;The MHC Sequencing Consortium 1999). A closer inspection of the G + C content reveals fairly uniform dispersion throughout the entire segment, although numerous high G + C content peaks (>50%) were locally detected, and in most cases associated with expressed genes and/or CpG islands, including recognition sites for rare CG cleavage enzymes ( Fig.…”