Using human artificial chromosomes to study centromere assembly and function

Abstract

Abstract:Centromeres are the site of assembly of the kinetochore, which directs chromosome segregation during cell division. Active centromeres are characterized by the presence of nucleosomes containing CENP-A and a specific chromatin environment that resembles that of active genes. Recent work using Human Artificial Chromosomes (HAC) sheds light on the fine balance of different histone post-translational modifications and transcription that exists at centromeres for kinetochore assembly and maintenance. Here, we review the use of HAC technology to understand centromere assembly and function. We put particular emphasis on studies using the alphoidtetO HAC, whose centromere can be specifically modified for epigenetic engineering studies. Author Comments:We suggest Professor Erich Nigg as an Editor (not found in the "Request Editor" list), who invited us to write this review.Response to Reviewers: Comments for the Author:Reviewer #1: Since there has been significant progress over the last few years with regards to HAC technology, particularly with alphoidtetO HACs, a review on this topic would be welcomed in the field, especially by these authors, who are world experts on Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporationthis topic. The review is mostly satisfying, but I would suggest two areas where the authors would be well served to make additions/changes to avoid disappointing readers hoping for more focus/organization/clarity. First, since the goal of this review is to "review the use of HAC technology to understand centromere assembly and function", it would be especially helpful if the authors summarized the evolution of HAC technology and the findings that came from targeting different tetR fusion proteins to the alphoidtetO HACs (probably most effectively in a table or schematic).We thank the reviewer for the thoughtful review of our MS. Following the reviewer´s advice, a table has been added explaining the findings that came from the alphoidtetO HAC studies in a timeline manner (Table 1).Second, more discussion on the pitfalls of current HACs would be desirable (currently only a couple sentences is mentioned in the future directions). From how it is written they sound so great that a reader would probably wonder why they are not used more widely in the research lab and/or clinic. There are problems with them, and it would be more clear and interesting, in my opinion, if more of the problems were laid out/challenges addressed.A description of limitations of the HAC technology have been added in the text, first limitations for HAC clinical use (p. 8-9) and additional limitations that should be overcome in the final remarks (p. 23). Some other changes that I'd suggest:1. It's confusing to refer to the same DNA as "alpha satellite", "α-satellite", and "alpha-satellite" throughout the review (most notably in the first two lines of the second paragraph). Why not just refer to it as "α-satellite" DNA?We changed the term to α-satellite DNA throughout the text to maintain consist...

Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription

“…Consistent with a global shut-down of transcription during prometaphase (the physiological equivalent of nocodazole-arrested cells), RNA Polymerase II is generally found displaced from chromatin ( Hsiung et al, 2016 ), with the notorious exception of the centromeric region ( Chan et al, 2012 ). Because recent studies have suggested that non-coding RNAs associate with centromere and kinetochore proteins, including CENP-A, CENP-C and Aurora B ( Blower, 2016 ; Carone et al, 2009 ; Du et al, 2010 ; Ferri et al, 2009 ; Molina et al, 2017 , 2016 ; Rošić et al, 2014 ; Wong et al, 2007 ), it is possible that transcription of non-coding RNAs plays a role in centromere assembly and function. However, whether this occurs during mitosis or during G1, when centromere assembly takes place in mammalian cells ( Jansen et al, 2007 ) remains unclear.…”

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission

“…The HAC loss rate is roughly 10-fold higher when compared with the native chromosomes (Nakano et al 2008), making the assay very sensitized and allowing a statistically significant number of events in a realistic sample size when studying the CIN phenotype in human cells. Previously, the HAC was used for low-throughput identification of drugs that elevate chromosome instability (CIN) in cancer cells (Lee et al 2013aKim et al 2016), as a gene delivery vector for the efficient and regulated expression of exogenous full-length genes in mammalian cells (Iida et al 2010;Kim et al 2011;Kouprina et al 2012Kouprina et al , 2013Kouprina et al , 2014Kononenko et al 2014;Liskovykh et al 2015;Lee et al 2018), and for studies of the epigenetic regulation of human kinetochores (Bergmann et al 2012;Ohzeki et al 2015;Molina et al 2017).…”

“…This assay is based on a nonessential human artificial chromosome (HAC) expressing a short half-life green fluorescent protein (GFP). This HAC, like other HACs, follows the rules of mitosis and chromosome segregation just like the natural chromosomes during the cell cycle progression (Nakano et al 2008;Bergmann et al 2012;Ohzeki et al 2015;Molina et al 2017). It is worth noting that the use of yeast artificial chromosomes (YACs) was critical for the discovery and systematic analysis of CIN genes in S. cerevisiae (Maine et al 1984;Spencer et al 1990;Kouprina et al 1993;Roberts et al 1994).…”

Mitotic progression, arrest, exit or death is determined by centromere integrity and independent of de novo transcription

“…Consistent with a global shut-down of transcription during prometaphase (the physiological equivalent of nocodazole-arrested cells), RNA Polymerase II is generally found displaced from chromatin (Hsiung et al, 2016), with the notorious exception of the centromeric region (Chan et al, 2012). Because recent studies have suggested that non-coding RNAs associate with centromere and kinetochore proteins, including CENP-A, CENP-C and Aurora B (Blower, 2016;Carone et al, 2009;Du et al, 2010;Ferri et al, 2009;Molina et al, 2017;Molina et al, 2016;Rošić et al, 2014;Wong et al, 2007), it remains possible that transcription of non-coding RNAs plays a role in centromere assembly and function. However, whether this occurs during mitosis or during G1, when centromere assembly takes place in mammalian cells (Jansen et al, 2007) is unclear.…”